Insulin Derivatives

ABSTRACT

The present invention is related to insulin derivatives having a side chain attached either to the α-amino group of the N-terminal amino acid residue of B chain or to an ε-amino group of a Lys residue present in the B chain of the parent insulin molecule via an amide bond which side chain comprises one or more residues of ethylenglycol, propyleneglycol and/or butyleneglycol containing independently at each termini a group selected from —NH 2  and —COOH; a fatty diacid moiety with 4 to 22 carbon atoms, at least one free carboxylic acid group or a group which is negatively charged at neutral pH; and possible linkers which link the individual components in the side chain together via amide or ether bonds, said linkers optionally comprising a free carboxylic acid group.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/814,374, filed Jul. 20, 2007, which is a 35 U.S.C. §371 nationalstage application of International Patent Application PCT/EP2006/050594(published as WO 2006/082205), filed Feb. 1, 2006, which claimedpriority of Danish Patent Application PA 2005 00156, filed Feb. 2, 2005;this application further claims priority under 35 U.S.C. §119 of U.S.Provisional Application 60/651,271, filed Feb. 8, 2005.

FIELD OF THE INVENTION

The present invention relates to novel human insulin derivatives whichare soluble at physiological pH values and have a prolonged profile ofaction. The invention also relates to methods of providing suchderivatives, to pharmaceutical compositions containing them, to a methodof treating diabetes and hyperglycaemia using the insulin derivatives ofthe invention and to the use of such insulin derivatives in thetreatment of diabetes and hyperglycaemia.

BACKGROUND OF THE INVENTION

Currently, the treatment of diabetes, both type 1 diabetes and type 2diabetes, relies to an increasing extent on the so-called intensiveinsulin treatment. According to this regimen, the patients are treatedwith multiple daily insulin injections comprising one or two dailyinjections of long acting insulin to cover the basal insulin requirementsupplemented by bolus injections of a rapid acting insulin to cover theinsulin requirement related to meals.

Long acting insulin compositions are well known in the art. Thus, onemain type of long acting insulin compositions comprises injectableaqueous suspensions of insulin crystals or amorphous insulin. In thesecompositions, the insulin compounds utilized typically are protamineinsulin, zinc insulin or protamine zinc insulin.

Certain drawbacks are associated with the use of insulin suspensions.Thus, in order to secure an accurate dosing, the insulin particles mustbe suspended homogeneously by gentle shaking before a defined volume ofthe suspension is withdrawn from a vial or expelled from a cartridge.Also, for the storage of insulin suspensions, the temperature must bekept within more narrow limits than for insulin solutions in order toavoid lump formation or coagulation.

Another type of long acting insulin compositions are solutions having apH value below physiological pH from which the insulin will precipitatebecause of the rise in the pH value when the solution is injected. Adrawback with these solutions is that the particle size distribution ofthe precipitate formed in the tissue on injection, and thus the releaseprofile of the medication, depends on the blood flow at the injectionsite and other parameters in a somewhat unpredictable manner. A furtherdrawback is that the solid particles of the insulin may act as a localirritant causing inflammation of the tissue at the site of injection.

Human insulin has three primary amino groups: the N-terminal group ofthe A-chain and of the B-chain and the ε-amino group of LysB29. Severalinsulin derivatives which are substituted in one or more of these groupsare known in the prior art. Thus, U.S. Pat. No. 3,528,960 (Eli Lilly)relates to N-carboxyaroyl insulins in which one, two or three primaryamino groups of the insulin molecule has a carboxyaroyl group.

According to GB Patent No. 1,492,997 (Nat. Res. Dev. Corp.), it has beenfound that insulin with a carbamyl substitution at N^(εB29) has animproved profile of hypoglycaemic effect.

JP laid-open patent application No. 1-254699 (Kodama Co., Ltd.)discloses insulin wherein a fatty acid is bound to the amino group ofPheB1 or to the ε-amino group of LysB29 or to both of these. The statedpurpose of the derivatisation is to obtain a pharmacologicallyacceptable, stable insulin preparation.

Insulins, which in the B30 position have an amino acid having at leastfive carbon atoms which cannot necessarily be coded for by a triplet ofnucleotides, are described in JP laid-open patent application No.57-067548 (Shionogi). The insulin analogues are claimed to be useful inthe treatment of diabetes mellitus, particularly in patients who areinsulin resistant due to generation of bovine or porcine insulinantibodies.

WO 95/07931 (Novo Nordisk A/S) discloses human insulin derivativeswherein the ε-amino group of LysB29 has a lipophilic substituent. Theseinsulin derivatives have a prolonged profile of action and are solubleat physiological pH values.

EP 894095 discloses insulin derivatives wherein the N-terminal group ofthe B-chain and/or the ε-amino group of Lys in position B28, B29 or B30has a substituent of the formula —CO—W—COOH where W can be a long chainhydrocarbon group. These insulin derivatives have a prolonged profile ofaction and are soluble at physiological pH values.

Unfortunately, many diabetics are unwilling to undertake intensivetherapy due to the discomfort associated with the many injectionsrequired to maintain close control of glucose levels. This type oftherapy can be both psychologically and physically painful. Upon oraladministration, insulin is rapidly degraded in the gastro intestinaltract and is not absorbed into the blood stream. Therefore, manyinvestigators have studied alternate routes for administering insulin,such as oral, rectal, transdermal, and nasal routes. Thus far, however,these routes of administration have not resulted in effective insulinabsorption.

Efficient pulmonary delivery of a protein is dependent on the ability todeliver the protein to the deep lung alveolar epithelium. Proteins thatare deposited in the upper airway epithelium are not absorbed to asignificant extent. This is due to the overlying mucus which isapproximately 30-40 μm thick and acts as a barrier to absorption. Inaddition, proteins deposited on this epithelium are cleared bymucociliary transport up the airways and then eliminated via thegastrointestinal tract. This mechanism also contributes substantially tothe low absorption of some protein particles. The extent to whichproteins are not absorbed and instead eliminated by these routes dependson their solubility, their size, as well as other less understoodcharacteristics.

It is however well recognised that the properties of peptides can beenhanced by grafting organic chain-like molecules onto them. Suchgrafting can improve pharmaceutical properties such as half life inserum, stability against proteolytical degradation, and reducedimmunogenicity.

The organic chain-like molecules often used to enhance properties arepolyethylene glycol-based or polyethylene based chains, i.e., chainsthat are based on the repeating unit —CH₂CH₂O—. Hereinafter, theabbreviation “PEG” is used for polyethyleneglycol.

Classical PEG technology takes advantage of providing polypeptides withincreased size (Stoke radius) by attaching a soluble organic molecule tothe polypeptide (Kochendoerfer, G., et al., Science (299) 884-, 2003).This technology leads to reduced clearance in man and animals of ahormone polypeptide compared to the native polypeptide. However thistechnique is often hampered by reduced potency of the hormonepolypeptides subjected to this technique (Hinds, K., et al.,Bioconjugate Chem. (11), 195-, 2000). WO 02/20033 discloses a generalmethod for the synthesis of well defined polymer modified peptides.

However, there is still a need for insulins having a more prolongedprofile of action than the insulin derivatives known up till now andwhich at the same time are soluble at physiological pH values and have apotency which is comparable to that of human insulin. Furthermore, thereis need for further insulin formulations which are well suited forpulmonary application.

SUMMARY OF THE INVENTION

The present invention is based on the recognition that acylation ofinsulin with one or more residues of ethylenglycol, propyleneglycoland/or butyleneglycol in combination with fatty diacid residues hassurprisingly shown a good bioavailability.

Organic chain-like molecules, which can be used to enhance properties,are poly-ethyleneglycol based, polypropyleneglycol based orpolybutyleneglycol based chains, i.e., chains that are based on therepeating unit CH₂CH₂O—, CH₂CH₂CH₂O— or CH₂CH₂CH₂CH₂O—. Hereinafter, theabbreviation “PEG” is used for polyethyleneglycol, “PPG” is used forpolypropyleneglycol and “PBG” is used for polybutyleneglycol.

In one aspect the present invention is related to insulin derivativeshaving a side chain attached either to the α-amino group of theN-terminal amino acid residue of the B chain or to an ε-amino group of aLys residue present in the B chain of the parent insulin molecule via anamide bond which side chain comprises one or more residues ofethylenglycol, propyleneglycol and/or butyleneglycol containingindependently at each termini a group selected from —NH₂ and —COOH; afatty diacid moiety with 4 to 22 carbon atoms; at least one freecarboxylic acid group or a group which is negatively charged at neutralpH; and possible linkers which link the individual components in theside chain together via amide, ether or amine bonds, said linkersoptionally comprising a free carboxylic acid group.

In one aspect the insulin derivatives contain a difunctional PEG, PPG orPBG group that has from 2 to 20; from 2 to 10 or from 2 to 5 residues ofethyleneglycol, propyleneglycol or butyleneglycol, respectively.

In one aspect the side chain of the insulin derivative comprise onesingle residue of ethyleneglycol.

In one aspect the side chain of the insulin derivative comprise onesingle residue of propyleneglycol.

In one aspect the side chain of the insulin derivative comprise onesingle residue of butyleneglycol.

In one aspect the side chain of the insulin derivative has singleresidues of ethylenglycol, propyleneglycol or butyleneglycol alone or incombination.

In one aspect the side chain of the insulin derivative has one residueof propyleneglycol and one residue of butyleneglycol.

In one aspect the fatty diacid comprises from 4 to 22 carbon atoms inthe carbon chain.

In one aspect the fatty diacid comprises from 6 to 22, from 8 to 20,from 8 to 18, from 4 to 18, from 6 to 18, from 8 to 16, from 8 to 22,from 8 to 17 or from 8 to 15 carbon atoms in the carbon chain.

In one aspect the linker is an amino acid residue, a peptide chain of2-4 amino acid residues or has the motif is α-Asp; β-Asp; α-Glu; δ-Glu;α-hGlu; δ-hGlu; —N(CH₂COOH)CH₂CO—; —N(CH₂CH₂COOH)CH₂CH₂CO—;—N(CH₂COOH)CH₂CH₂CO— or —N(CH₂CH₂COOH)CH₂CO—.

In one aspect the Lys residue in the B chain will be position B3, B29 orin one of positions B23-B30.

In another aspect the invention is related to an insulin derivativehaving the formula

wherein Ins is the parent insulin moiety which via the α-amino group ofthe N-terminal amino acid residue of the B chain or an ε-amino group ofa Lys residue present in the B chain of the insulin moiety is bound tothe CO— group in the side chain via an amide bond; each n isindependently 0, 1, 2, 3, 4, 5 or 6;

-   Q₁, Q₂, Q₃, and Q₄ independently of each other can be    -   (CH₂CH₂O)_(s)—; (CH₂CH₂CH₂O)_(s)—; (CH₂CH₂CH₂CH₂O)_(s)—;        (CH₂CH₂OCH₂CH₂CH₂CH₂O)_(s)— or (CH₂CH₂CH₂OCH₂CH₂CH₂CH₂O)_(s)—        where s is 1-20    -   —(CH₂)_(r)— where r is an integer from 4 to 22; or a divalent        hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a        number of —CH₂— groups sufficient to give a total number of        carbon atoms in the chain in the range of 4 to 22;    -   —(CH₂)_(t)— or —(CH₂OCH₂)_(t)—, where t is an integer from 1 to        6;    -   —(CR₁R₂)_(q)—, where R₁ and R₂ independently of each other can        be H, —COOH, (CH₂)₁₋₆COOH and R₁ and R₂ can be different at each        carbon, and q is 1-6,    -   —((CR₃R₄)_(q1))₁—(NHCO—(CR₃R₄)_(q1)—NHCO)₁₋₂—((CR₃R₄)_(q1))₁ or        —((CR₃R₄)_(q1))₁—(CONH—(CR₃R₄)_(q1)—CONH)₁₋₂—((CR₃R₄)_(q1)—)—,        —((CR₃R₄)_(q1))₁—(NHCO—(CR₃R₄)_(q1)—CONH)₁₋₂—((CR₃R₄)_(q1))₁ or        —((CR₃R₄)_(q1))₁—(CONH—(CR₃R₄)_(q1)—NHCO)₁₋₂—((CR₃R₄)_(q1))₁        where R₃ and R₄ independently of each other can be H, —COOH, and        R₃ and R₄ can be different at each carbon, and q₁ is 1-6-, or    -   a bond;

with the proviso that Q₁-Q₄ are different;

X₁, X₂ and X₃ are independently

-   -   O;    -   a bond; or

where R is hydrogen or —(CH₂)_(p)—COOH, —(CH₂)_(p)—SO₃H,—(CH₂)_(p)—PO₃H₂, —(CH₂)_(p)—O—SO₃H; —(CH₂)_(p)—O—PO₃H₂; or—(CH₂)_(p)-tetrazol-5-yl, where each p independently of the other p's isan integer in the range of 1 to 6; and

Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)₂,

—N(CH₂COOH)₂;

—SO₃H

—OSO₃H

—OPO3H₂

—PO₃H₂ or

-tetrazol-5-yl

and any Zn²⁺ complex thereof.

Where mentioned that R₁, R₂, R₃ and R₄ can be different at each carbonis meant that R₁, R₂, R₃ and R₄ can be different for each value of q orq₁.

In one aspect r is from 6 to 22, from 8 to 20, from 8 to 18, from 4 to18, from 6 to 18 from 8 to 16 from 8 to 22 from 8 to 17 from 8 to 15.

In another aspect s is in the range of 2-12, 2-4 or 2-3.

In another aspect s is 1.

In one aspect n is from 1-6, from 2-6, from 2-5, from 2-4, from 0-2 orfrom 2-3.

In one aspect q is from 1-5, from1-4, from 1-3 or from 1-2.

In one aspect q₁ is from 1-5, from 1-4, from 1-3 or from 1-2.

In one aspect t is from 1-6, from 1-5, from 1-4, from 1-3 or from 1-2.

In one aspect Z is —COOH.

In one aspect Z is —CO-Asp.

In another aspect Z is —CO-Glu.

In another aspect Z is —CO-Gly.

In another aspect Z is —CO-Sar.

In another aspect Z is —CH(COOH)₂.

In another aspect Z is —N(CH₂COOH)₂.

In another aspect Z is —SO₃H.

In another aspect Z is —PO₃H.

In another aspect Z is O—SO₃H;

In another aspect Z is O—PO₃H₂;

In another aspect Z is tetrazol-5-yl.

In a further aspect the parent insulin is a desB30 human insulinanalogue.

Non limiting examples of parent insulins are human insulin; desB1 humaninsulin; desB30 human insulin; GlyA21 human insulin; GlyA21 desB30 humaninsulin; AspB28 human insulin; porcine insulin; LysB28 ProB29 humaninsulin; GlyA21 ArgB31 ArgB32 human insulin; LysB3 GIuB29 human insulinor AspB28 desB30 human insulin.

In a still further aspect the insulin derivative are selected from thegroup consisting ofN^(εB29)-(3-[2-{2-(2-[ω-carboxy-pentadecanoyl-γ-glutamyl-(2-amino-ethoxy)]-ethoxy)-ethoxy}-ethoxy]-propinoyl)desB30 human insulin,N^(εB29)-(3-[2-{2-(2-[ω-carboxy-heptadecanoyl-δ-glutamyl-(2-amino-ethoxy)]-ethoxy)-ethoxyl-ethoxy]-propinoyl)desB30 human insulin,N^(ε629)-{3-[2-(2-{2-[2-(ω-carboxy-pentadecanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionyl-γ-glutamyldesB30 human insulin,N^(εB29)-(ω-[2-(2-{2-[2-(2-carboxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethylcarbamoyl]-heptadecanoyl-α-glutamyl)desB30 human insulin,N^(εB29)-(ω-[2-(2-{2-[2-(2-carboxy-ethoxy)-ethoxy]-ethoxyl-ethoxy)-ethylcarbamoyl]-heptadecanoyl-γ-glutamyl)desB30 human insulin,N^(εB29)-3-[2-(2-{2-[2-(ω-carboxy-heptadecanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionyl-γ-glutamyldesB30 human insulin,N^(εB29)-(3-(3-{2-[2-(3-[7-carboxyheptanoylamino]propoxy)ethoxy]-ethoxy}propylcarbamoyl)propionyl)desB30 human insulin,N^(εB29)-(3-(3-{4-[3-(7-Carboxyheptanoylamino)propoxy]butoxy}propylcarbamoyl)-propionyl-γ-glutamyl)desB30 human insulin,N^(εB29)-(3-(3-{2-[2-(3-[9-Carboxynonanoylamino]propoxy)ethoxy]ethoxy}-propylcarbamoyl)propionyl)desB30 human insulin,N^(εB29)-(3-(2-{2-[2-(9-carboxynonanoylamino)ethoxy]ethoxy}ethylcarbamoyl)propionyl-γ-glutamyl)desB30 human insulin,N^(εB29)-(3-(3-{4-[3-(9-Carboxynonanoylamino)propoxy]butoxy}-propylcarbamoyl)propionyl-γ-glutamyl)desB30 human insulin,N^(εB29)-(2-[3-(2-(2-{2-(7-carboxyheptanoylamino)ethoxy}ethoxy)-ethylcarbamoyl]propionyl-γ-glutamyl)desB30 human insulin,N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxypentadecanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]propionyl))desB30 human insulin,N^(εB29)-(3-(2-{2-[2-(2-{2-[2-(2-{2-[2-(2-{2-[2-(ω-carboxy-tridecanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionoyl-γ-glutamyl)desB30 human insulin,N^(εB29)-(3-[2-(2-{2-[2-(ω-Carboxy-tridecanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionoyl-γ-glutamyl)desB30 human insulin,N^(εB29)-(3-[2-(2-{2-[2-(2-{2-[2-(ω-carboxy-tridecanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-propionoyl-γ-glutamyl)desB30 human insulin,N^(εB29)-(3-(2-{2-[2-(ω-Carboxy-pentadecanoylamino)-ethoxy]-ethoxy}-ethylcarbamoyl)-propionyl-γ-glutamyl)desB30 human insulin,N^(εB29)-(3-(3-{2-[2-(3-[ω-Carboxypentadecanoylamino]propoxy)ethoxy]-ethoxy}propylcarbamoyl)propionyl-γ-glutamyl)desB30 human insulin,N^(εB29)-(3-(3-{4-[3-(ω-Carboxyundecanoylamino)propoxy]butoxypropylcarbamoyl)propionyl-γ-glutamyl)desB30 human insulin,N^(εB29)-(3-(3-{4-[3-(ω-carboxytridecanoylamino)propoxy]butoxypropyl-carbamoyl)propionyl-γ-glutamyl)desB30 human insulin,N^(εB29)-(3-(2-{2-[2-(ω-Carboxyundecanoylamino)ethoxy]ethoxy}ethylcarbamoyl)propionyl-γ-glutamyl)desB30 human insulin,N^(εB29)-(3-(2-{2-[2-(ω-carboxytridecanoylamino)ethoxy]ethoxy}ethylcarbamoyl)-propionyl-γ-glutamyl)desB30 human insulin,N^(εB29)-{3-[2-(2-{2-[2-(ω-carboxy-pentadecanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]propionyl-gamma-γ-D-glutamyl)desB30 human insulin,N^(εB29)-{3-[2-(2-{2-[2-(7-carboxyheptanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]-propionyl-γ-glutamyl}desB30 human insulin,N^(εB29)-{3-[2-(2-{2-[2-(9-carboxynonanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]propionyl-γ-glutamyl}desB30 human insulin,N^(εB29)-{3-[2-(2-{2-[2-(ω-carboxyundecanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]-propionyl-γ-glutamyl}desB30 human insulin,N^(εB29)-{3-[2-(2-{2-[2-(ω-carboxytridecanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]propionyl-γ-glutamyl}desB30 human insulin.

Insulin derivatives according to the invention may be provided in theform of essentially zinc free compounds or in the form of zinccomplexes. When zinc complexes of an insulin derivative according to theinvention are provided, two Zn²⁺ ions, three Zn²⁺ ions or four Zn²⁺ ionscan be bound to each insulin hexamer. Solutions of zinc complexes of theinsulin derivatives will contain mixtures of such species.

In a further aspect the invention is related to a pharmaceuticalcomposition comprising a therapeutically effective amount of an insulinderivative according to the invention together with a pharmaceuticallyacceptable carrier can be provided for the treatment of type 1 diabetes,type 2 diabetes and other states that cause hyperglycaemia in patientsin need of such a treatment. An insulin derivative according to theinvention can be used for the manufacture of a pharmaceuticalcomposition for use in the treatment of type 1 diabetes, type 2 diabetesand other states that cause hyperglycaemia.

In a further aspect of the invention, there is provided a pharmaceuticalcomposition for treating type 1 diabetes, type 2 diabetes and otherstates that cause hyperglycaemia in a patient in need of such atreatment, comprising a therapeutically effective amount of an insulinderivative according to the invention in mixture with an insulin or aninsulin analogue which has a rapid onset of action, together withpharmaceutically acceptable carriers and additives.

In a further aspect the invention is related to a pulmonary applicationfor treating type 1 diabetes, type 2 diabetes and other states thatcause hyperglycaemia in a patient in need of such a treatment,comprising a therapeutically effective amount of an insulin derivativeaccording to the invention optionally in mixture with an insulin or aninsulin analogue which has a rapid onset of action, together withpharmaceutically acceptable carriers and additives.

In one aspect the invention provides a pharmaceutical composition beinga mixture of an insulin derivative according to the invention and arapid acting insulin analogue selected group consisting of AspB28 humaninsulin; LysB28 ProB29 human insulin and LysB3 GluB29 human insulin.

The insulin derivative according to the invention and the rapid actinginsulin analogue can be mixed in a ratio from about 90/10%; about 70/30%or about 50/50%.

In a further aspect of the invention, there is provided a method oftreating type 1 diabetes, type 2 diabetes and other states that causehyperglycaemia in a patient in need of such a treatment, comprisingadministering to the patient a therapeutically effective amount of aninsulin derivative according to the invention together with apharmaceutically acceptable carrier and pharmaceutical acceptableadditives.

In a further aspect of the invention, there is provided a method oftreating type 1 diabetes, type 2 diabetes and other states that causehyperglycaemia in a patient in need of such a treatment, comprisingadministering to the patient a therapeutically effective amount of aninsulin derivative according to the invention in mixture with an insulinor an insulin analogue which has a rapid onset of action, together witha pharmaceutically acceptable carrier and pharmaceutical acceptableadditives.

In another aspect of the invention the insulin derivatives has a sidechain attached either to the α-amino group of the N-terminal amino acidresidue of B chain or to an ε-amino group of a Lys residue present inthe B chain of the parent insulin molecule via an amide bond which sidechain comprises a monodisperse, diffunctionel PEG group containingindependently at each termini a group selected from —OH; —NH₂ and —COOH;a fatty diacid moiety with 4 to 22 carbon atoms, at least one freecarboxylic acid group or a group which is negatively charged at neutralpH; and possible linkers which link the individual components in theside chain together via amide, ether or amine bonds, said linkersoptionally comprising a free carboxylic acid group.

In another aspect of the invention the PEG group of the insulinderivative has from 1 to 20; from 1 to 10 or from 1 to 5 ethyleneresidues.

In another aspect of the invention the insulin derivatives has a sidechain attached either to the α-amino group of the N-terminal amino acidresidue of B chain or to an ε-amino group of a Lys residue present inthe B chain of the parent insulin molecule via an amide bond which sidechain comprises a monodisperse, diffunctionel PEG group containingindependently at each termini a group selected from —OH; —NH₂ and —COOH;a fatty diacid moiety with 4 to 22 carbon atoms, at least one freecarboxylic acid group or a group which is negatively charged at neutralpH; and possible linkers which link the individual components in theside chain together via amide, ether or amine bonds, said linkersoptionally comprising a free carboxylic acid group.

In a further aspect of the invention the insulin derivatives comprises adifunctionel PEG group which has from 1 to 20; from 1 to 10 or from 1 to5 ethylene units.

In a further aspect of the invention the insulin derivatives comprises afatty diacid which comprises from 4 to 22 carbon atoms in the carbonchain.

In a further aspect of the invention the insulin derivatives comprises afatty acid, wherein the fatty diacid comprises from 6 to 22, from 8 to20, from 8 to 18, from 4 to 18, from 6 to 18, from 8 to 16, from 8 to22, from 8 to 17 or from 8 to 15 carbon atoms in the carbon chain.

In a further aspect of the invention the insulin derivatives comprises alinker wherein the linker is an amino acid residue, a peptide chain of2-4 amino acid residues or has the motif α-Asp, β-Asp, α-Glu, γ-Glu,α-hGlu, δ-hGlu, —N(CH₂COOH)CH₂CO—, —N(CH₂CH₂COOH)CH₂CH₂CO—,—N(CH₂COOH)CH₂CH₂CO— or —N(CH₂CH₂COOH)CH₂CO—

In a further aspect of the invention the insulin derivatives comprises aLys residue wherein the Lys residue in the B chain of the parent insulinin in either position B3 or in one of positions B23-30.

In a further aspect of the invention the insulin derivatives has theformula

wherein lns is the parent insulin moiety which via the α-amino group ofthe N-terminal amino acid residue of the B chain or an ε-amino group ofa Lys residue present in the B chain of the insulin moiety is bound tothe CO— group in the side chain via an amide bond;

-   each n is independently 0, 1, 2, 3, 4, 5 or 6;-   Q₁, Q₂, Q₃, and Q₄ independently of each other can be    -   (CH₂CH₂O)_(s)— where s is 1-20,    -   —(CH₂)_(r)— where r is an integer from 4 to 22; or a divalent        hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a        number of —CH₂— groups sufficient to give a total number of        carbon atoms in the chain in the range of 4 to 22;    -   —(CH₂)_(t)— or —(CH₂OCH₂)_(t)—, where t is an integer from 1 to        6;    -   —(CR₁R₂)_(q)—, where R₁ and R₂ independently of each other can        be H, —COOH, and R₁ and R₂ can be different at each carbon, and        q is 1-6,    -   —((CR₃R₄)_(q1))₁—(NHCO—(CR₃R₄)_(q1)—NHCO)₁₋₂—((CR₃R₄)_(q1))₁ or        —((CR₃R₄)_(q1))₁—(CONH—(CR₃R₄)_(q1)—CONH)₁₋₂—((CR₃R₄)_(q1)—,        where R₃ and R₄ independently of each other can be H, —COOH, and        R₃ and R₄ can be different at each carbon, and q₁ is 1-6-, or    -   a bond;

with the proviso that Q₁-Q₄ are different;

X and V and G are independently

-   -   O;    -   a bond; or

where R is hydrogen or —(CH₂)_(p)—COOH, —(CH₂)_(p)—SO₃H,—(CH₂)_(p)—PO₃H₂, —(CH₂)_(p)—O—SO₃H; —(CH₂)_(p)—O—PO₃H₂; or—(CH₂)_(p)-tetrazolyl, where each p independently of the other p's is aninteger in the range of 1 to 6; and

Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)₂,

—N(CH₂COOH)₂;

—SO₃H

—OSO₃H

—OPO3H₂

—PO₃H₂ or

-tetrazolyl.

In a further aspect of the invention the insulin derivatives accordingto the formula, s is from 6 to 22, from 8 to 20, from 8 to 18, from 4 to18, from 6 to 18, from 8 to 16, from 8 to 22, from 8 to 17 or from 8 to15.

In a further aspect of the invention the insulin derivatives accordingto the formula s is from 1-20, from 1-10 or from 1-5.

In a further aspect of the invention the insulin derivative according tothe formula, Z is —COOH.

In a further aspect of the invention the insulin derivative according tothe invention, the parent insulin is a desB30 human insulin analogue.

In a further aspect of the invention the insulin derivative according tothe invention, the parent insulin is selected from the group consistingof human insulin; desB1 human insulin; desB30 human insulin; GlyA21human insulin; GlyA21 desB30human insulin; AspB28 human insulin; porcineinsulin; LysB28ProB29 human insulin; GlyA21ArgB31ArgB32 human insulin;and LysB3GluB29 human insulin.

In a further aspect of the invention the insulin derivative accordingthe invention is selected from the group consisting ofN^(εB29)—(N^(α)—(HOOC(CH₂)₁₄CO)-γ-L-Glu-HN(CH₂CH₂O)₄CH₂CH₂CO) desB30human insulin;N^(εB29)—(N^(α)—(HOOC(CH₂)₁₆CO)-γ-L-Glu-HN(CH₂CH₂O)₄CH₂CH₂CO) des(B30)human insulin;N^(εB29)—(N^(α)—(HOOC(CH₂CH₂O)₄CH₂CH₂NH—OC(CH₂)₁₆CO)-α-L-Glu-) des(B30)human insulin;N^(εB29)-{3-[2-(2-{2-[2-(15-Carboxy-pentadecanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionyl-gamma-GludesB30 insulin; andN^(εB29)-3-[2-(2-{2-[2-(17-Carboxy-heptadecanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionylγ-Glu desB30 insulin

In a further aspect of the invention there is provided a pharmaceuticalcomposition for the treatment of diabetes in a patient in need of suchtreatment, comprising a therapeutically effective amount of an insulinderivative according to the invention together with a pharmaceuticallyacceptable carrier.

In a further aspect of the invention there is provided a pharmaceuticalcomposition for the treatment of diabetes in a patient in need of suchtreatment, comprising a therapeutically effective amount of an insulinderivative according to the invention in mixture with an insulin or aninsulin analogue which has a rapid onset of action, together with apharmaceutically acceptable carrier.

In a further aspect of the invention there is provided a pharmaceuticalcomposition according to the invention intended for pulmonaladministration.

In a further aspect of the invention there is provided a method oftreating diabetes in a patient in need of such a treatment, comprisingadministering to the patient a therapeutically effective amount of aninsulin derivative according to claim 1 together with a pharmaceuticallyacceptable carrier.

In a further aspect of the invention there is provided a method oftreating diabetes in a patient in need of such a treatment, comprisingadministering to the patient a therapeutically effective amount of aninsulin derivative according to claim 1 in mixture with an insulin or aninsulin analogue which has a rapid onset of action, together with apharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION

The present insulin derivatives are characterized by having a side chainattached to a Lys group in the B chain or to the N-terminal amino groupin the B-chain of the parent insulin molecule which side chain comprisesone or more residues of ethylenglycol, propylene-glycol and/orbutyleneglycol and a fatty diacid moiety.

The insulin derivative according to the invention is furthermorecharacterized in having at least one free carboxylic acid group in theside chain and may comprise up to 2, 3 or 4 free carboxylic acid groupsor a group which is negatively charged at neutral pH.

The insulin derivatives will only contain one lysine residue. Thislysine residue may either be in position B29 as in human insulin or inone of position B3, B30 or B23 to B28.

The residues of ethylenglycol, propyleneglycol and/or butyleneglycolwill have any combination of the three groups —OH; —NH₂ and —COOH ateach end. The residues of ethylenglycol, propyleneglycol and/orbutyleneglycol will typically be in the form of an ethyleneglycolresidue followed by a butyleneglycol residue or have a chain length of 2to 20 PEG, PPG or PBG residues corresponding to a molecular weight ofabout 200 to 800.

The residues of ethylenglycol, propyleneglycol and/or butyleneglycolwill typically be in the form of an ethyleneglycol residue followed by abutylen residue —(CH₂CH₂OCH₂CH₂CH₂CH₂O)_(m) where m is 1 to 20.

The difunctional PEG or PPG or PBG will have any combination of thethree groups —OH; —NH₂, and —COOH at each end and will typically have achain length of 1 to 20 PEG residues corresponding to a molecular weightof about 200 to 1000.

Non limiting examples of amino PEG moieties areH₂N—(CH₂)_(u)—(OCH₂CH₂)_(m)—O(CH₂)_(u)—COOH andH₂N—(CH₂)_(v)—NH—CO—(CH₂)_(u)—(OCH₂CH₂)_(m)—(CH₂)_(u)—COOH, where u areindependently 1 to 6, m is 2 to 20 and v is 1 to 6.

Non limiting examples of amino PPG moieties areH₂N—(CH₂)_(u)—(OCH₂CH₂CH₂)_(m)—O(CH₂)_(u)—COOH andH₂N—(CH₂)_(v)—NH—CO—(CH₂)_(u)—(OCH₂CH₂CH₂)_(m)—(CH₂)_(u)—COOH, where uare independently 1 to 6, m is 2 to 20 and v is 1 to 6.

Non limiting examples of amino PBG moieties areH₂N—(CH₂)_(u)—(OCH₂CH₂CH₂CH₂)_(m)—O(CH₂)_(u)—COOH andH₂N—(CH₂)_(v)—NH—CO—(CH₂)_(u)—(OCH₂CH₂CH₂CH₂)_(m)—(CH₂)_(u)—COOH, whereu are independently 1 to 6, m is 2 to 20 and v is 1 to 6.

The fatty diacid will typically comprise from 4 to 22, from 6 to 22,from 8 to 20, from 8 to 18, from 4 to 18, from 6 to 18, from 8 to 16,from 8 to 22, from 8 to 12, from 8 to 10, from 8 to 17 or from 8 to 15carbon atoms in the carbon chain.

Non limiting examples of the fatty diacid moiety are diacids with theformula HOOC—(CH₂)_(r1)—COOH, where r1 is 4 to 22. Examples of fattydiacids are succinic acid, hexanedioic acid, octanedioic acid,decanedioic acid, dodecanedioic acid, tetradecanedioic acid,hexadecanedioic acid or octadecandedioic acid.

The insulin moiety—in the present text also referred to as the parentinsulin—or insulin derivative according to the invention can be anaturally occurring insulin such as human insulin or porcine insulin.Alternatively, the parent insulin can be an insulin analogue.

In one group of parent insulin analogues, the amino acid residue atposition A21 is Asn.

In another group of parent insulin analogues, the amino acid residue atposition B1 has been deleted. A specific example from this group ofparent insulin analogues is desB1 human insulin.

In another group of parent insulin analogues, the amino acid residue atposition B30 has been deleted. A specific example from this group ofparent insulin analogues is desB30 human insulin.

In another group of parent insulin analogues, the amino acid residue atposition B28 is Asp. A specific example from this group of parentinsulin analogues is AspB28 human insulin.

In another group of parent insulin analogues, the amino acid residue atposition B28 is Lys and the amino acid residue at position B29 is Pro. Aspecific example from this group of parent insulin analogues is LysB28ProB29 human insulin.

In another group of parent insulin analogues the amino acid residue inposition B30 is Lys and the amino acid residue in position B29 is anycodable amino acid except Cys, Arg and Lys. An example is an insulinanalogue where the amino acid residue at position B29 is Thr and theamino acid residue at position B30 is Lys. A specific example from thisgroup of parent insulin analogues is ThrB29 LysB30 human insulin.

In another group of parent insulin analogues, the amino acid residue atposition B3 is Lys and the amino acid residue at position B29 is Glu. Aspecific example from this group of parent insulin analogues is LysB3GluB29 human insulin.

The linkers will typically be an amino acid residue or a chain of aminoacid residue comprising up to four amino acids. Thus, the linker may beselected from the group consisting of α-Asp; β-Asp; α-Glu; γ-Glu;α-hGlu; δ-hGlu; —N(CH₂COOH)CH₂CO—, —N(CH₂CH₂COOH)CH₂CH₂CO—;—N(CH₂COOH)CH₂CH₂CO— or —N(CH₂CH₂COOH)CH₂CO—

In a further aspect the linker can be a chain composed of two amino acidresidues of which one has from 4 to 10 carbon atoms and a carboxylicacid group in the side chain while the other has from 2 to 11 carbonatoms but no free carboxylic acid group. The amino acid residue with nofree carboxylic acid group can be a neutral, codable α-amino acidresidue. Examples of such linkers are are: α-Asp-Gly; Gly-α-Asp;β-Asp-Gly; Gly-β-Asp; α-Glu-Gly; Gly-α-Glu; γ-Glu-Gly; Gly-γ-Glu;α-hGlu-Gly; Gly-α-hGlu; δ-hGlu-Gly; and Gly-δ-hGlu.

In a further aspect the linker is a chain composed of two amino acidresidues, independently having from 4 to 10 carbon atoms, and bothhaving a carboxylic acid group in the side chain. Examples of suchlinkers are: α-Asp-α-Asp; α-Asp-α-Glu; α-Asp-α-hGlu; α-Asp-β-Asp;α-Asp-γ-Glu; α-Asp-δ-hGlu; β-Asp-α-Asp; β-Asp-α-Glu; β-Asp-α-hGlu;β-Asp-β-Asp; β-Asp-γ-Glu; β-Asp-δ-hGlu; α-Glu-α-Asp; α-Glu-α-Glu;α-Glu-α-hGlu; α-Glu-β-Asp; α-Glu-δ-Glu; α-Glu-δ-hGlu; γ-Glu-α-Asp;γ-Glu-α-Glu; γ-Glu-α-hGlu; γ-Glu-β-Asp; γ-Glu-γ-Glu; γ-Glu-δ-hGlu;α-hGlu-α-Asp; α-hGlu-α-Glu; α-hGlu-α-hGlu; α-hGlu-β-Asp; α-hGlu-γ-Glu;α-hGlu-δ-hGlu; δ-hGlu-α-Asp; δ-hGlu-α-Glu; δ-hGlu-α-hGlu; δ-hGlu-β-Asp;δ-hGlu-γ-Glu; and δ-hGlu-δ-hGlu.

In a further aspect the linker is a chain composed of three amino acidresidues, independently having from 4 to 10 carbon atoms, the amino acidresidues of the chain being selected from the group of residues having aneutral side chain and residues having a carboxylic acid group in theside chain so that the chain has at least one residue which has acarboxylic acid group in the side chain. In one aspect, the amino acidresidues are codable residues.

In a further aspect, the linker is a chain composed of four amino acidresidues, independently having from 4 to 10 carbon atoms, the amino acidresidues of the chain being selected from the group having a neutralside chain and residues having a carboxylic acid group in the side chainso that the chain has at least one residue which has a carboxylic acidgroup in the side chain. In one aspect, the amino acid residues arecodable residues.

Examples of insulin derivatives according to the invention are thefollowing compounds:

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxyheptadecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-γ-glutamyl)desB30 human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxyhexadecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-γ-glutamyl)desB30 human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxytridecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-γ-glutamyl)desB30 human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxyundecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-γ-glutamyl)desB30 human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxynonanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-γ-glutamyl)desB30 human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxyheptanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-γ-glutamyl)desB30 human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxyheptadecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl)desB30 human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxyhexadecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl)desB30 human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxytridecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl)desB30 human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxyundecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl)desB30 human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxynonanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl)desB30 human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxyheptanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl)desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyheptadecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyhexadecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxypentadecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxytridecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyundecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxynonanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyheptanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyheptadecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyhexadecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxypentadecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxytridecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyundecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxynonanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyheptanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxypentadecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl)desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyhexadecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl)desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxypentadecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl)desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxytridecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl)desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyundecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl)desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxynonanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl)desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyheptanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl)desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyheptadecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl-γ-glutamyl)desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyhexadecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl-γ-glutamyl)desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxypentadecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl-γ-glutamyl)desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxytridecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl-γ-glutamyl)desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyundecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl-γ-glutamyl)desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxynonanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl-γ-glutamyl)desB30 human insulin

N^(εB29)-(3-(2-{2-[2-(ω-carboxyheptanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl-γ-glutamyl)desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyheptadecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxypentadecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxypentadecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxytridecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyundecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxynonanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyheptanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyheptadecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyhexadecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxypentadecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxytridecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyundecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxynonanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyheptanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)desB30 human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxyheptadecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-γ-glutamyl)human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxyhexadecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-γ-glutamyl)human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxytridecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-γ-glutamyl)human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxyundecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-γ-glutamyl)human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxynonanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-γ-glutamyl)human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxyheptanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-γ-glutamyl)human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxyheptadecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl)human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxyhexadecanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]propionyl)human insulin

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxytridecanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]propionyl)human insulin

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxyundecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl)human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxynonanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl)human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxyheptanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl)human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyheptadecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyhexadecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxypentadecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxytridecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyundecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxynonanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyheptanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyheptadecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyhexadecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxypentadecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxytridecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyundecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxynonanoylamino)propoxy]ethoxy}propylcarbamoyl)propionyl-γ-glutamyl)human insulin

N^(εB29)-(3-(3-{2-[3-(ω-carboxyheptanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxypentadecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl)human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyhexadecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl)human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxypentadecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl)human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxytridecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl)human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyundecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl)human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxynonanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl)human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyheptanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl)human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyheptadecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl-γ-glutamyl)human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyhexadecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl-γ-glutamyl)human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxypentadecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl-γ-glutamyl)human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxytridecanoylamino)ethoxy]ethoxy}ethylcarbamoyl)-propionyl-γ-glutamyl)human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyundecanoylamino)ethoxy]ethoxy}ethylcarbamoyl)propionyl-γ-glutamyl)human insulin

N^(εB29)-(3-(2-{2-[2-(ω-carboxynonanoylamino)ethoxy]ethoxy}ethylcarbamoyl)-propionyl-γ-glutamyl)human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyheptanoylamino)ethoxy]ethoxy}ethylcarbamoyl)-propionyl-γ-glutamyl)human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyheptadecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxypentadecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxypentadecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxytridecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyundecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxynonanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyheptanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyheptadecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyhexadecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxypentadecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxytridecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyundecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxynonanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyheptanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)human insulin:

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxyheptadecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxyhexadecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxytridecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxyundecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxynonanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxyheptanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxyheptadecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl)B28D human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxyhexadecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl)B28D human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxytridecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl)B28D human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxyundecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl)B28D human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxynonanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl)B28D human insulin;

N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxyheptanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl)B28D human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyheptadecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)B28D human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyhexadecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)B28D human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxypentadecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)B28D human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxytridecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)B28D human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyundecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)B28D human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxynonanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)B28D human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyheptanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)B28D human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyheptadecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyhexadecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxypentadecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxytridecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyundecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxynonanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyheptanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxypentadecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl)B28D human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyhexadecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl)B28D human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxypentadecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl)B28D human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxytridecanoylamino)ethoxy]ethoxy}ethylcarbamoyl)-propionyl)B28D human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyundecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl)B28D human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxynonanoylamino)ethoxy]ethoxy}ethylcarbamoyl)-propionyl)B28D human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyheptanoylamino)ethoxy]ethoxy}ethylcarbamoyl)-propionyl)B28D human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyheptadecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyhexadecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxypentadecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxytridecanoylamino)ethoxy]ethoxy}ethylcarbamoyl)-propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyundecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxynonanoylamino)ethoxy]ethoxy}ethylcarbamoyl)-propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyheptanoylamino)ethoxy]ethoxy}ethylcarbamoyl)-propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyheptadecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxypentadecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxypentadecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxytridecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyundecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxynonanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyheptanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)B28D human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyheptadecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)B28D human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyhexadecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)B28D human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxypentadecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)B28D human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxytridecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)B28D human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyundecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)B28D human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxynonanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)B28D human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyheptanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)B28D human insulin;

N^(εB29)-(3-[3-(2-{2-[2-(ω-carboxyheptadecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-γ-glutamyl)B28D, desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[2-(ω-carboxyhexadecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-γ-glutamyl)B28D, desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[2-(ω-carboxytridecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-γ-glutamyl)B28D, desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[2-(ω-carboxyundecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-γ-glutamyl)B28D, desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[2-(ω-carboxynonanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl-γ-glutamyl)B28D, desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[2-(ω-carboxyheptanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]-propionyl-γ-glutamyl)B28D, desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[2-(ω-carboxyheptadecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl)B28D, desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[2-(ω-carboxyhexadecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl)B28D, desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[2-(ω-carboxytridecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl)B28D, desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[2-(ω-carboxyundecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl)B28D, desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[2-(ω-carboxynonanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl)B28D, desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[2-(ω-carboxyheptanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl)B28D, desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyheptadecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)B28D, desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyhexadecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)B28D, desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxypentadecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)B28D, desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxytridecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)B28D, desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyundecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)B28D, desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxynonanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)B28D, desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyheptanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl)B28D, desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyheptadecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)B28D, desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyhexadecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)B28D, desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxypentadecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)B28D, desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxytridecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)B28D, desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyundecanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)B28D, desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxynonanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)B28D, desB30 human insulin;

N^(εB29)-(3-(3-{2-[3-(ω-carboxyheptanoylamino)propoxy]ethoxy}-propylcarbamoyl)propionyl-γ-glutamyl)B28D, desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxypentadecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl)B28D, desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyhexadecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl)B28D, desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxypentadecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl)B28D, desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxytridecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl)B28D, desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyundecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl)B28D, desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxynonanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl)B28D, desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyheptanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl)B28D, desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyheptadecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl-γ-glutamyl)B28D, desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyhexadecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl-γ-glutamyl)B28D, desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxypentadecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl-γ-glutamyl)B28D, desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxytridecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl-γ-glutamyl)B28D, desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyundecanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl-γ-glutamyl)B28D, desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxynonanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl-γ-glutamyl)B28D, desB30 human insulin;

N^(εB29)-(3-(2-{2-[2-(ω-carboxyheptanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionyl-γ-glutamyl)B28D, desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyheptadecanoylamino)propoxy]ethoxy}-ethoxy)propylcarbamoyl]propionyl-γ-glutamyl)B28D, desB30 human insulin

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxypentadecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)B28D, desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxypentadecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)B28D, desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxytridecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)B28D, desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyundecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)B28D, desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxynonanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)B28D, desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyheptanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl-γ-glutamyl)B28D, desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyheptadecanoylamino)propoxy]ethoxy}-ethoxy)propylcarbamoyl]propionyl)B28D, desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyhexadecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)B28D, desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxypentadecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)B28D, desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxytridecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)B28D, desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyundecanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)B28D, desB30 human insulin;

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxynonanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)B28D, desB30 human insulin; and

N^(εB29)-(3-[3-(2-{2-[3-(ω-carboxyheptanoylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]propionyl)B28D, desB30 human insulin

Representative formulas are:

In a further aspect, the present invention relates to insulinderivatives which have an overall hydrophobicity which is essentiallysimilar to that of human insulin.

In a further aspect, the insulin derivatives of the present inventionhave a hydrophobic index, k′rel, which is in the range from about 0.02to about 10, from about 0.1 to about 5; from about 0.5 to about 5; fromabout 0.2 to about 2; from about 0.2 to about 1; from about 0.1 to about2; or from about 0.5 to about 2.

According to one aspect of the present invention, the insulinderivatives will comprise a side chain of general formula (I) as definedabove which have at least one free carboxylic acid group and accordingto a further aspect, the side chain will optionally hold one or morefree carboxylic acid groups.

The hydrophobicity (hydrophobic index) of the insulin derivatives of theinvention relative to human insulin, k′_(rel), was measured on aLiChrosorb RP18 (5 μm, 250×4 mm) HPLC column by isocratic elution at 40°C. using mixtures of A) 0.1 M sodium phosphate buffer, pH 7.3,containing 10% acetonitrile, and B) 50% acetonitrile in water aseluents. The elution was monitored by following the UV absorption of theeluate at 214 nm. Void time, t₀, was found by injecting 0.1 mM sodiumnitrate. Retention time for human insulin, t_(human), was adjusted to atleast 2t₀ by varying the ratio between the A and B solutions.k′_(rel)=(t_(derivative)−t₀)/(t_(human)−t₀).

In another aspect, the invention relates to a pharmaceutical compositioncomprising an insulin derivative according to the invention which issoluble at physiological pH values.

In another aspect, the invention relates to a pharmaceutical compositioncomprising an insulin derivative according to the invention which issoluble at pH values in the interval from about 6.5 to about 8.5.

In another aspect, the invention relates to a pharmaceutical compositionwith a prolonged profile of action which comprises an insulin derivativeaccording to the invention.

In another aspect, the invention relates to a pharmaceutical compositionwhich is a solution containing from about 120 nmol/ml to about 2400nmol/ml, from about 400 nmol/ml to about 2400 nmol/ml, from about 400nmol/ml to about 1200 nmol/ml, from about 600 nmol/ml to about 2400nmol/ml, or from about 600 nmol/ml to about 1200 nmol/ml of an insulinderivative according to the invention or of a mixture of the insulinderivative according to the invention with a rapid acting insulinanalogue.

The starting product for the acylation, the parent insulin or insulinanalogue or a precursor thereof can be produced by either well-knowpeptide synthesis or by well known recombinant production in suitabletransformed microorganisms. Thus the insulin starting product can beproduced by a method which comprises culturing a host cell containing aDNA sequence encoding the polypeptide and capable of expressing thepolypeptide in a suitable nutrient medium under conditions permittingthe expression of the peptide, after which the resulting peptide isrecovered from the culture.

As an example desB30 human insulin can be produced from a human insulinprecursor B(1-29)-Ala-Ala-Lys-A(1-21) which is produced in yeast asdisclosed in U.S. Pat. No. 4,916,212. This insulin precursor can then beconverted into desB30 human insulin by ALP cleavage of the Ala-Ala-Lyspeptide chain to give desB30 human insulin which can then be acylated togive the present insulintives.

The medium used to culture the cells may be any conventional mediumsuitable for growing the host cells, such as minimal or complex mediacontaining appropriate supplements. Suitable media are available fromcommercial suppliers or may be prepared according to published recipes(e.g. in catalogues of the American Type Culture Collection). Thepeptide produced by the cells may then be recovered from the culturemedium by conventional procedures including separating the host cellsfrom the medium by centrifugation or filtration, precipitating theproteinaceous components of the supernatant or filtrate by means of asalt, e.g. ammonium sulphate, purification by a variety ofchromatographic procedures, e.g. ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like,dependent on the type of peptide in question.

The DNA sequence encoding the parent insulin may suitably be of genomicor cDNA origin, for instance obtained by preparing a genomic or cDNAlibrary and screening for DNA sequences coding for all or part of thepolypeptide by hybridisation using synthetic oligonucleotide probes inaccordance with standard techniques (see, for example, Sambrook, J,Fritsch, E F and Maniatis, T, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, New York, 1989). The DNA sequenceencoding the parent insulin may also be prepared synthetically byestablished standard methods, e.g. the phosphoamidite method describedby Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869, orthe method described by Matthes et al., EMBO Journal 3 (1984), 801-805.The DNA sequence may also be prepared by polymerase chain reaction usingspecific primers, for instance as described in U.S. Pat. No. 4,683,202or Saiki et al., Science 239 (1988), 487-491.

The DNA sequence may be inserted into any vector which may convenientlybe subjected to recombinant DNA procedures, and the choice of vectorwill often depend on the host cell into which it is to be introduced.Thus, the vector may be an autonomously replicating vector, i.e. avector which exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g. a plasmid.Alternatively, the vector may be one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector in which the DNA sequenceencoding the parent insulin is operably linked to additional segmentsrequired for transcription of the DNA, such as a promoter. The promotermay be any DNA sequence which shows transcriptional activity in the hostcell of choice and may be derived from genes encoding proteins eitherhomologous or heterologous to the host cell. Examples of suitablepromoters for directing the transcription of the DNA encoding the parentinsulin in a variety of host cells are well known in the art, cf. forinstance Sambrook et al., supra.

The DNA sequence encoding the parent insulin may also, if necessary, beoperably connected to a suitable terminator, polyadenylation signals,transcriptional enhancer sequences, and translational enhancersequences. The recombinant vector of the invention may further comprisea DNA sequence enabling the vector to replicate in the host cell inquestion.

The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell or one whichconfers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin,chloramphenicol, neomycin, hygromycin or methotrexate.

To direct a peptide of the present invention into the secretory pathwayof the host cells, a secretory signal sequence (also known as a leadersequence, prepro sequence or pre sequence) may be provided in therecombinant vector. The secretory signal sequence is joined to the DNAsequence encoding the peptide in the correct reading frame. Secretorysignal sequences are commonly positioned 5′ to the DNA sequence encodingthe peptide. The secretory signal sequence may be that normallyassociated with the peptide or may be from a gene encoding anothersecreted protein.

The procedures used to ligate the DNA sequences coding for the parentinsulin, the promoter and optionally the terminator and/or secretorysignal sequence, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambrook et al., supra).

The host cell into which the DNA sequence or the recombinant vector isintroduced may be any cell which is capable of producing the presentpeptide and includes bacteria, yeast, fungi and higher eukaryotic cells.Examples of suitable host cells well known and used in the art are,without limitation, E. coli, Saccharomyces cerevisiae, or mammalian BHKor CHO cell lines.

The parent insulin molecule is then converted into the insulinderivatives of the invention by introducing of the relevant side chainin either the B1 position or in the chosen Lys position in the B-chain.The side chain can be introduced by any convenient method and manymethods are disclosed in the prior art for acylation of an amino group.More details will appear from the following examples.

Pharmaceutical Compositions

The insulin derivatives of this invention of the claimed formula can,for example, be administered subcutaneously, orally, or pulmonary.

For subcutaneous administration, the compounds of the formula areformulated analogously with the formulation of known insulins.Furthermore, for subcutaneous administration, the compounds of theformula are administered analogously with the administration of knowninsulins and, generally, the physicians are familiar with thisprocedure.

The insulin derivatives of this invention may be administered byinhalation in a dose effective manner to increase circulating insulinlevels and/or to lower circulating glucose levels. Such administrationcan be effective for treating disorders such as diabetes orhyperglycemia. Achieving effective doses of insulin requiresadministration of an inhaled dose of insulin derivative of thisinvention of more than about 0.5 μg/kg to about 50 μg/kg. Atherapeutically effective amount can be determined by a knowledgeablepractitioner, who will take into account factors including insulinlevel, blood glucose levels, the physical condition of the patient, thepatient's pulmonary status, or the like.

According to the invention, insulin derivative of this invention may bedelivered by inhalation to achieve prolonged duration of action.Administration by inhalation can result in pharmacokinetics comparableto subcutaneous administration of insulins. Different inhalation devicestypically provide similar pharmacokinetics when similar particle sizesand similar levels of lung deposition are compared.

According to the invention, insulin derivative of this invention may bedelivered by any of a variety of inhalation devices known in the art foradministration of a therapeutic agent by inhalation. These devicesinclude metered dose inhalers, nebulizers, dry powder generators,sprayers, and the like. Preferably, insulin derivative of this inventionis delivered by a dry powder inhaler or a sprayer. There are a severaldesirable features of an inhalation device for administering insulinderivative of this invention. For example, delivery by the inhalationdevice is advantageously reliable, reproducible, and accurate. Theinhalation device should deliver small particles, for example, less thanabout 10 μm, for example about 1-5 μm, for good respirability. Somespecific examples of commercially available inhalation devices suitablefor the practice of this invention are Turbohaler™ (Astra), Rotahaler®(Glaxo), Diskus® (Glaxo), Spiros™ inhaler (Dura), devices marketed byInhale Therapeutics, AERx™ (Aradigm), the Ultravent® nebulizer(Mallinckrodt), the Acorn II® nebulizer (Marquest Medical Products), theVentolin® metered dose inhaler (Glaxo), the Spinhaler® powder inhaler(Fisons), or the like.

As those skilled in the art will recognize, the formulation of insulinderivative of this invention, the quantity of the formulation delivered,and the duration of administration of a single dose depend on the typeof inhalation device employed. For some aerosol delivery systems, suchas nebulizers, the frequency of administration and length of time forwhich the system is activated will depend mainly on the concentration ofinsulin conjugate in the aerosol. For example, shorter periods ofadministration can be used at higher concentrations of insulin conjugatein the nebulizer solution. Devices such as metered dose inhalers canproduce higher aerosol concentrations, and can be operated for shorterperiods to deliver the desired amount of insulin conjugate. Devices suchas powder inhalers deliver active agent until a given charge of agent isexpelled from the device. In this type of inhaler, the amount of insulinderivative of this invention in a given quantity of the powderdetermines the dose delivered in a single administration.

The particle size of insulin derivative of this invention in theformulation delivered by the inhalation device is critical with respectto the ability of insulin to make it into the lungs, and preferably intothe lower airways or alveoli. Preferably, the insulin derivative of thisinvention is formulated so that at least about 10% of the insulinconjugate delivered is deposited in the lung, preferably about 10 toabout 20%, or more. It is known that the maximum efficiency of pulmonarydeposition for mouth breathing humans is obtained with particle sizes ofabout 2 μm to about 3 μm. When particle sizes are above about 5 μmpulmonary deposition decreases substantially. Particle sizes below about1 μm cause pulmonary deposition to decrease, and it becomes difficult todeliver particles with sufficient mass to be therapeutically effective.Thus, particles of the insulin derivative delivered by inhalation have aparticle size preferably less than about 10 μm, more preferably in therange of about 1 μm to about 5 μm. The formulation of the insulinderivative is selected to yield the desired particle size in the choseninhalation device.

Advantageously for administration as a dry powder, an insulin derivativeof this invention is prepared in a particulate form with a particle sizeof less than about 10 μm, preferably about 1 to about 5 μm. Thepreferred particle size is effective for delivery to the alveoli of thepatient's lung. Preferably, the dry powder is largely composed ofparticles produced so that a majority of the particles have a size inthe desired range. Advantageously, at least about 50% of the dry powderis made of particles having a diameter less than about 10 μm. Suchformulations can be achieved by spray drying, milling, or critical pointcondensation of a solution containing insulin conjugate and otherdesired ingredients. Other methods also suitable for generatingparticles useful in the current invention are known in the art.

The particles are usually separated from a dry powder formulation in acontainer and then transported into the lung of a patient via a carrierair stream. Typically, in current dry powder inhalers, the force forbreaking up the solid is provided solely by the patient's inhalation. Inanother type of inhaler, air flow generated by the patient's inhalationactivates an impeller motor which deagglomerates the particles.

Formulations of insulin derivatives of this invention for administrationfrom a dry powder inhaler typically include a finely divided dry powdercontaining the derivative, but the powder can also include a bulkingagent, carrier, excipient, another additive, or the like. Additives canbe included in a dry powder formulation of insulin conjugate, forexample, to dilute the powder as required for delivery from theparticular powder inhaler, to facilitate processing of the formulation,to provide advantageous powder properties to the formulation, tofacilitate dispersion of the powder from the inhalation device, tostabilize the formulation (for example, antioxidants or buffers), toprovide taste to the formulation, or the like. Advantageously, theadditive does not adversely affect the patient's airways. The insulinderivative can be mixed with an additive at a molecular level or thesolid formulation can include particles of the insulin conjugate mixedwith or coated on particles of the additive. Typical additives includemono-, di-, and polysaccharides; sugar alcohols and other polyols, suchas, for example, lactose, glucose, raffinose, melezitose, lactitol,maltitol, trehalose, sucrose, mannitol, starch, or combinations thereof;surfactants, such as sorbitols, diphosphatidyl choline, or lecithin; orthe like. Typically an additive, such as a bulking agent, is present inan amount effective for a purpose described above, often at about 50% toabout 90% by weight of the formulation. Additional agents known in theart for formulation of a protein such as insulin analogue protein canalso be included in the formulation.

A spray including the insulin derivatives of this invention can beproduced by forcing a suspension or solution of insulin conjugatethrough a nozzle under pressure. The nozzle size and configuration, theapplied pressure, and the liquid feed rate can be chosen to achieve thedesired output and particle size. An electrospray can be produced, forexample, by an electric field in connection with a capillary or nozzlefeed. Advantageously, particles of insulin conjugate delivered by asprayer have a particle size less than about 10 μm, preferably in therange of about 1 μm to about 5 μm.

Formulations of insulin derivatives of this invention suitable for usewith a sprayer will typically include the insulin derivative in anaqueous solution at a concentration of about 1 mg to about 20 mg ofinsulin conjugate per ml of solution. The formulation can include agentssuch as an excipient, a buffer, an isotonicity agent, a preservative, asurfactant, and, preferably, zinc. The formulation can also include anexcipient or agent for stabilization of the insulin derivative, such asa buffer, a reducing agent, a bulk protein, or a carbohydrate. Bulkproteins useful in formulating insulin conjugates include albumin,protamine, or the like. Typical carbohydrates useful in formulatinginsulin conjugates include sucrose, mannitol, lactose, trehalose,glucose, or the like. The insulin derivative formulation can alsoinclude a surfactant, which can reduce or prevent surface-inducedaggregation of the insulin conjugate caused by atomization of thesolution in forming an aerosol. Various conventional surfactants can beemployed, such as polyoxyethylene fatty acid esters and alcohols, andpolyoxyethylene sorbitol fatty acid esters. Amounts will generally rangebetween about 0.001 and about 4% by weight of the formulation.

Pharmaceutical compositions containing an insulin derivative accordingto the present invention may also be administered parenterally topatients in need of such a treatment. Parenteral administration may beperformed by subcutaneous, intramuscular or intravenous injection bymeans of a syringe, optionally a pen-like syringe. Alternatively,parenteral administration can be performed by means of an infusion pump.Further options are to administer the insulin nasally or pulmonally,preferably in compositions, powders or liquids, specifically designedfor the purpose.

Injectable compositions of the insulin derivatives of the invention canbe prepared using the conventional techniques of the pharmaceuticalindustry which involve dissolving and mixing the ingredients asappropriate to give the desired end product. Thus, according to oneprocedure, an insulin derivative according to the invention is dissolvedin an amount of water which is somewhat less than the final volume ofthe composition to be prepared. An isotonic agent, a preservative and abuffer is added as required and the pH value of the solution isadjusted—if necessary—using an acid, e.g. hydrochloric acid, or a base,e.g. aqueous sodium hydroxide as needed. Finally, the volume of thesolution is adjusted with water to give the desired concentration of theingredients.

In a further aspect of the invention the buffer is selected from thegroup consisting of sodium acetate, sodium carbonate, citrate,glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogenphosphate, disodium hydrogen phosphate, sodium phosphate, andtris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate,maleic acid, fumaric acid, tartaric acid, aspartic acid or mixturesthereof. Each one of these specific buffers constitutes an alternativeaspect of the invention.

In a further aspect of the invention the formulation further comprises apharmaceutically acceptable preservative which may be selected from thegroup consisting of phenol, o-cresol, m-cresol, p-cresol, methylp-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butylp-hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, andthiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodiumdehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethoniumchloride, chlorphenesine (3p-chlorphenoxypropane-1,2-diol) or mixturesthereof. In a further aspect of the invention the preservative ispresent in a concentration from 0.1 mg/ml to 20 mg/ml. In a furtheraspect of the invention the preservative is present in a concentrationfrom 0.1 mg/ml to 5 mg/ml. In a further aspect of the invention thepreservative is present in a concentration from 5 mg/ml to 10 mg/ml. Ina further aspect of the invention the preservative is present in aconcentration from 10 mg/ml to 20 mg/ml. Each one of these specificpreservatives constitutes an alternative aspect of the invention. Theuse of a preservative in pharmaceutical compositions is well-known tothe skilled person. For convenience reference is made to Remington: TheScience and Practice of Pharmacy, 19^(th) edition, 1995.

In a further aspect of the invention the formulation further comprisesan isotonic agent which may be selected from the group consisting of asalt (e.g. sodium chloride), a sugar or sugar alcohol, an amino acid(e.g. L-glycine, L-histidine, arginine, lysine, isoleucine, asparticacid, tryptophan, threonine), an alditol (e.g. glycerol (glycerine),1,2-propanediol (propyleneglycol), 1,3-propanediol, 1,3-butanediol)polyethyleneglycol (e.g. PEG400), or mixtures thereof. Any sugar such asmono-, di-, or polysaccharides, or water-soluble glucans, including forexample fructose, glucose, mannose, sorbose, xylose, maltose, lactose,sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, solublestarch, hydroxyethyl starch and carboxymethylcellulose-Na may be used.In one aspect the sugar additive is sucrose. Sugar alcohol is defined asa C4-C8 hydrocarbon having at least one —OH group and includes, forexample, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol,and arabitol. In one aspect the sugar alcohol additive is mannitol. Thesugars or sugar alcohols mentioned above may be used individually or incombination. There is no fixed limit to the amount used, as long as thesugar or sugar alcohol is soluble in the liquid preparation and does notadversely effect the stabilizing effects achieved using the methods ofthe invention. In one aspect, the sugar or sugar alcohol concentrationis between about 1 mg/ml and about 150 mg/ml. In a further aspect of theinvention the isotonic agent is present in a concentration from 1 mg/mlto 50 mg/ml. In a further aspect of the invention the isotonic agent ispresent in a concentration from 1 mg/ml to 7 mg/ml. In a further aspectof the invention the isotonic agent is present in a concentration from 8mg/ml to 24 mg/ml. In a further aspect of the invention the isotonicagent is present in a concentration from 25 mg/ml to 50 mg/ml. Each oneof these specific isotonic agents constitutes an alternative aspect ofthe invention. The use of an isotonic agent in pharmaceuticalcompositions is well-known to the skilled person. For conveniencereference is made to Remington: The Science and Practice of Pharmacy,19^(th) edition, 1995.

Typical isotonic agents are sodium chloride, mannitol, dimethyl sulfoneand glycerol and typical preservatives are phenol, m-cresol, methylp-hydroxybenzoate and benzyl alcohol.

Examples of suitable buffers are sodium acetate, glycylglycine, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and sodiumphosphate.

A composition for nasal administration of an insulin derivativeaccording to the present invention may, for example, be prepared asdescribed in European Patent No. 272097 (to Novo Nordisk A/S).

Compositions containing insulin derivatives of this invention can beused in the treatment of states which are sensitive to insulin. Thus,they can be used in the treatment of type 1 diabetes, type 2 diabetesand hyperglycaemia for example as sometimes seen in seriously injuredpersons and persons who have undergone major surgery. The optimal doselevel for any patient will depend on a variety of factors including theefficacy of the specific insulin derivative employed, the age, bodyweight, physical activity, and diet of the patient, on a possiblecombination with other drugs, and on the severity of the state to betreated. It is recommended that the daily dosage of the insulinderivative of this invention be determined for each individual patientby those skilled in the art in a similar way as for known insulincompositions.

Where expedient, the insulin derivatives of this invention may be usedin mixture with other types of insulin, e.g. insulin analogues with amore rapid onset of action. Examples of such insulin analogues aredescribed e.g. in the European patent applications having thepublication Nos. EP 214826 (Novo Nordisk A/S), EP 375437 (Novo NordiskA/S) and EP 383472 (Eli Lilly & Co.).

In a further aspect of the present invention the present compounds areadministered in combination with one or more further active substancesin any suitable ratios. Such further active agents may be selected fromantidiabetic agents, antihyperlipidemic agents, antiobesity agents,antihypertensive agents and agents for the treatment of complicationsresulting from or associated with diabetes.

Suitable antidiabetic agents include insulin, GLP-1 (glucagon likepeptide-1) derivatives such as those disclosed in WO 98/08871 (NovoNordisk A/S), which is incorporated herein by reference, as well asorally active hypoglycemic agents.

Suitable orally active hypoglycemic agents preferably includeimidazolines, sulfonylureas, biguanides, meglitinides,oxadiazolidinediones, thiazolidinediones, insulin sensitizers,α-glucosidase inhibitors, agents acting on the ATP-dependent potassiumchannel of the pancreatic β-cells eg potassium channel openers such asthose disclosed in WO 97/26265, WO 99/03861 and WO 00/37474 (NovoNordisk A/S) which are incorporated herein by reference, potassiumchannel openers, such as ormitiglinide, potassium channel blockers suchas nateglinide or BTS-67582, glucagon antagonists such as thosedisclosed in WO 99/01423 and WO 00/39088 (Novo Nordisk A/S and AgouronPharmaceuticals, Inc.), all of which are incorporated herein byreference, GLP-1 agonists such as those disclosed in WO 00/42026 (NovoNordisk A/S and Agouron Pharmaceuticals, Inc.), which are incorporatedherein by reference, DPP-IV (dipeptidyl peptidase-IV) inhibitors, PTPase(protein tyrosine phosphatase) inhibitors, inhibitors of hepatic enzymesinvolved in stimulation of gluconeogenesis and/or glycogenolysis,glucose uptake modulators, GSK-3 (glycogen synthase kinase-3)inhibitors, compounds modifying the lipid metabolism such asantihyperlipidemic agents and antilipidemic agents, compounds loweringfood intake, and PPAR (peroxisome proliferator-activated receptor) andRXR (retinoid X receptor) agonists such as ALRT-268, LG-1268 or LG-1069.

Definitions

With “desB30 insulin”, “desB30 human insulin” is meant a natural insulinor an analogue thereof lacking the B30 amino acid residue. Similarly,“desB29desB30 insulin or “desB29desB30 human insulin” means a naturalinsulin or an analogue thereof lacking the B29 and B30 amino acidresidues.

With “B(1-29)” is meant a natural insulin B chain or an analogue thereoflacking the B30 amino acid residue. “A(1-21)” means the natural insulinA chain or an analogue thereof.

With “B1”, “A1” etc. is meant the amino acid residue in position 1 inthe B chain of insulin (counted from the N-terminal end) and the aminoacid residue in position 1 in the A chain of insulin (counted from theN-terminal end), respectively. The amino acid residue in a specificposition may also be denoted as e.g. Phe^(B1) which means that the aminoacid residue in position B1 is a phenylalanine residue.

With “Insulin” as used herein is meant human insulin with disulfidebridges between Cys^(A7) and Cys^(B7) and between Cys^(A20) andCys^(B19) and an internal disulfide bridge between Cys^(A6) andCys^(A11), porcine insulin and bovine insulin.

By “insulin analogue” as used herein is meant a polypeptide which has amolecular structure which formally can be derived from the structure ofa naturally occurring insulin, for example that of human insulin, bydeleting and/or substituting at least one amino acid residue occurringin the natural insulin and/or by adding at least one amino acid residue.The added and/or substituted amino acid residues can either be codableamino acid residues or other naturally occurring amino acid residues orpurely synthetic amino acid residues.

The insulin analogues may be such wherein position 28 of the B chain maybe modified from the natural Pro residue to one of Asp, Lys, or Ile. Inanother aspect Lys at position B29 is modified to Pro. In one aspect B30may be Lys and then B29 can be any codable amino acid except Cys, Met,Arg and Lys. Also, Asn at position A21 may be modified to Ala, Gln, Glu,Gly, His, Ile, Leu, Met, Ser, Thr, Trp, Tyr or Val, in particular toGly, Ala, Ser, or Thr and preferably to Gly. Furthermore, Asn atposition B3 may be modified to Lys or Asp. Further examples of insulinanalogues are desB30 human insulin, desB30 human insulin analogues;insulin analogues wherein one or both of B1 and B2 have been deleted;insulin analogues wherein the A-chain and/or the B-chain have anN-terminal extension and insulin analogues wherein the A-chain and/orthe B-chain have a C-terminal extension. Further insulin analogues aresuch wherein. Thus one or two Arg may be added to position B1. Also oneor more of B26-B30 may have been deleted

By “insulin derivative” as used herein is meant a naturally occurringinsulin or an insulin analogue which has been chemically modified, e.g.by introducing a side chain in one or more positions of the insulinbackbone or by oxidizing or reducing groups of the amino acid residuesin the insulin or by converting a free carboxylic group to an estergroup or acylating a free amino group or a hydroxy group.

The expression “a codable amino acid” or “a codable amino acid residue”is used to indicate an amino acid or amino acid residue which can becoded for by a triplet (“codon”) of nucleotides.

α-Asp is the L-form of —HNCH(CO—)CH₂COOH.

β-Asp is the L-form of —HNCH(COOH)CH₂CO—.

α-Glu is the L-form of —HNCH(CO—)CH₂CH₂COOH.

γ-Glu is the L-form of —HNCH(COOH)CH₂CH₂CO—.

The expression “an amino acid residue having a carboxylic acid group inthe side chain” designates amino acid residues like Asp, Glu and hGlu.The amino acids can be in either the L- or D-configuration. If nothingis specified it is understood that the amino acid residue is in the Lconfiguration.

The expression “an amino acid residue having a neutral side chain”designates amino acid residues like Gly, Ala, Val, Leu, Ile, Phe, Pro,Ser, Thr, Cys, Met, Tyr, Asn and Gln.

When an insulin derivative according to the invention is stated to be“soluble at physiological pH values” it means that the insulinderivative can be used for preparing insulin compositions that are fullydissolved at physiological pH values. Such favourable solubility mayeither be due to the inherent properties of the insulin derivative aloneor a result of a favourable interaction between the insulin derivativeand one or more ingredients contained in the vehicle.

The following abbreviations have been used in the specification andexamples:

Aad: Alpha-amino-adipic acid (homoglutamic acid)

Bzl=Bn: benzyl

CN: Alpha-cyano-4-hydroxycinnamic acid

DIEA: N, N-diisopropylethylamine

DMF: N,N-dimethylformamide

IDA: Iminodiacetic acid

Sar: Sarcosine (N-methyl-glycine)

tBu: tert-butyl

TSTU: O-(N-succinimidyI)-1,1,3,3-tetramethyluronium tetrafluoroborate

THF: Tetrahydrofuran

EtOAc: Ethyl acetate

DIPEA: N, N-Diisopropylethylamine

HOAt: 1-Hydroxy-7-azabenzotriazole

TEA: Triethyl amine

SA: Sinapic acid

Su: succinimidyl=2,5-dioxo-pyrrolidin-1-yl

TFA: Trifluoracetic acid

DCM: Dichloromethane

DMSO: Dimethyl sulphoxide

PEG: Polyethyleneglycol

PBG: Poly-1,4-butyleneglycol

PPG: Poly-1,3-propyleneglycol

TLC: Thin Layer Chromatography

RT: Room temperature

With “fatty diacid” is meant a linear or branched dicarboxylic acidshaving at least 2 carbon atoms and being saturated or unsaturated. Nonlimiting examples of fatty diacids are succinic acid, hexanedioic acid,octanedioic acid, decanedioic acid, dodecanedioic acid, tetradecanedioicacid, hexadecanedioic acid and octadecanedioic acid.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference in theirentirety and to the same extent as if each reference were individuallyand specifically indicated to be incorporated by reference and were setforth in its entirety herein (to the maximum extent permitted by law).

All headings and sub-headings are used herein for convenience only andshould not be construed as limiting the invention in any way.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

The citation and incorporation of patent documents herein is done forconvenience only and does not reflect any view of the validity,patentability, and/or enforceability of such patent documents.

This invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw.

Examples

The following examples and general procedures refer to intermediatecompounds and final products identified in the specification and in thesynthesis schemes. The preparation of the compounds of the presentinvention is described in detail using the following examples, but thechemical reactions described are disclosed in terms of their generalapplicability to the preparation of compounds of the invention.Occasionally, the reaction may not be applicable as described to eachcompound included within the disclosed scope of the invention. Thecompounds for which this occurs will be readily recognised by thoseskilled in the art. In these cases the reactions can be successfullyperformed by conventional modifications known to those skilled in theart, that is, by appropriate protection of interfering groups, bychanging to other conventional reagents, or by routine modification ofreaction conditions. Alternatively, other reactions disclosed herein orotherwise conventional will be applicable to the preparation of thecorresponding compounds of the invention. In all preparative methods,all starting materials are known or may easily be prepared from knownstarting materials. All temperatures are set forth in degrees Celsiusand unless otherwise indicated, all parts and percentages are by weightwhen referring to yields and all parts are by volume when referring tosolvents and eluents.

The compounds of the invention can be purified by employing one or moreof the following procedures which are typical within the art. Theseprocedures can—if needed—be modified with regard to gradients, pH,salts, concentrations, flow, columns and so forth. Depending on factorssuch as impurity profile, solubility of the insulins in questionetcetera, these modifications can readily be recognised and made by aperson skilled in the art.

After acidic HPLC or desalting, the compounds are isolated bylyophilisation of the pure fractions.

After neutral HPLC or anion exchange chromatography, the compounds aredesalted, precipitated at isoelectrical pH, or purified by acidic HPLC.

Typical Purification Procedures:

The HPLC system is a Gilson system consisting of the following: Model215 Liquid handler, Model 322-H2 Pump and a Model 155 UV Dector.Detection is typically at 210 nm and 280 nm.

The Akta Purifier FPLC system (Amersham Biosciences) consists of thefollowing: Model P-900 Pump, Model UV-900 UV detector, Model pH/C-900 pHand conductivity detector, Model Frac-950 Frction collector. UVdetection is typically at 214 nm, 254 nm and 276 nm.

Acidic HPLC:

Column: Macherey-Nagel SP 250/21 Nucleusil 300-7 C4 Flow: 8 ml/minBuffer A: 0.1% TFA in acetonitrile Buffer B: 0.1% TFA in water.Gradient: 0.0-5.0 min: 10% A 5.00-30.0 min: 10% A to 90% A 30.0-35.0min: 90% A 35.0-40.0 min: 100% A

Neutral HPLC:

Column: Phenomenex, Jupiter, C4 5 μm 250 × 10.00 mm, 300 Å Flow: 6ml/min Buffer A: 5 mM TRIS, 7.5 mM (NH₄)₂SO₄, pH = 7.3, 20% CH₃CN BufferB: 60% CH3CN, 40% water Gradient: 0-5 min: 10% B 5-35 min: 10-60% B35-39 min: 60% B 39-40 min: 70% B 40-43.5 min: 70% B

Anion Exchange Chromatography:

00Column: RessourceQ, 1 ml Flow: 6 ml/min Buffer A: 0.09% NH₄HCO₃, 0.25%NH₄OAc, 42.5% ethanol pH 8.4 Buffer B: 0.09% NH₄HCO₃, 2.5% NH₄OAc, 42.5%ethanol pH 8.4 Gradient: 100% A to 100% B during 30 column volumes

Desalting:

Column: HiPrep 26/10 Flow: 10 ml/min, 6 column volumes Buffer: 10 mMNH₄HCO₃

Analytical Procedures:

Method 1:

Two Waters 510 HPLC pumps Waters 2487 Dual λ Absorbance detector BufferA: 0.1% TFA in acetonitrile. Buffer B: 0.1% TFA in water. Flow: 1.5ml/min. Gradient: 1-17 min: 25% B to 85% B, 17-22 min: 85% B, 22-23 min:85% B to 25% B, 23-30 min 25% B, 30-31 min 25% B flow: 0.15 ml/min.Column: C4 5μ 150 × 4.60 mm Phenomenex (Jupiter). Detection: UV 214 nm.

Method 2:

Two Waters 510 HPLC pumps Waters 2487 Dual λ Absorbance detector BufferA: 0.1% TFA, 10% CH₃CN, 89.9% water. Buffer B: 0.1% TFA, 80% CH₃CN,19.9% water. Flow: 1.5 ml/min. Gradient: 0-17 min: 20%-90% B, 17-21 min90% B. Column: C4 5μ 150 × 4.60 mm Phenomenex (Jupiter), kept at 40° C.Detection: UV 214 nm.

Method 3:Two Waters 510 HPLC Pumps

Waters 486 Tunable Absorbance Detector Waters 717 Autosampler Column: C45μ 150 × 4_60 mm Phenomenex (Jupiter). Injection: 20 μl. Buffer A: 80%0.0125 M Tris, 0.0187 M (NH₄)₂SO₄ pH = 7, 20% CH₃CN. Buffer B: 80%CH₃CN, 20% water. Flow: 1.5 ml/min. Gradient: 0 min 5% B -> 20 min 55% B-> 22 min 80% B -> 24 min 80% B -> 25 min 5% B 32 min 5% B. Detection:UV 214 nm.

Method 4:

Two Waters 510 HPLC pumps Waters 2487 Dual λ Absorbance detector Column:C4 5μ 150 × 4.60 mm Phenomenex (Jupiter). Injection: 20 μl Buffer A: 80%0.0125 M Tris, 0.0187 M (NH₄)₂SO₄ pH = 7, 20% CH₃CN Buffer B: 80% CH₃CN,20% water Flow: 1.5 ml/min Gradient: 0 min 10% B -> 20 min 50% B -> 22min 60% B -> 23 min 10% B -> 30 min 10% B -> 31 min 10% B flow 0.15 minDetection: 214 nm

Method 5:

Waters 2695 separations module Waters 996 Photodiode Array DetectorColumn: C4 5μ 150 × 4.60 mm Phenomenex (Jupiter). Injection: 25 μlBuffer A: 80% 0.01 M Tris, 0.015 M (NH₄)₂SO₄ pH = 7.3; 20% CH₃CN BufferB: 20% water; 80% CH₃CN Flow: 1.5 ml/min Gradient: 1-20 min: 5-50% B,20-22 min: 50-60% B, 22-23 min: 60-5% B, 23-30 min 0% B 30-31 min 0-5%B, flow: 0.15 ml/min. Detection: 214 nm

Method 6:

Waters 2795 Separations Module

Waters 2996 Photodiode Array Detector

Waters Micromass ZQ 4000 Electrospray Mass Spectrometer

LC-Method:

Column: Phenomenex, Jupiter 5μ C4 300 Å 50 × 4.60 mm Buffer A: 0.1% TFAin water Buffer B: CH₃CN Flow: 1 ml/min Gradient: 0-7.5 min: 10-90% B7.5-8.5 min: 90-10% B 8.5-9.5 min 10% B 9.5-10.00 min 10% B, flow: 0.1ml/min

MS method: Mw: 500-2000 ES+

-   -   Cone Voltage 60V    -   Scantime 1    -   Interscan delay: 0.1

Method 7:

Agilent 1100 series Column: GraceVydac Protein C4, 5um 4.6 × 250 mm(Cat# 214TP54) Buffer A: 10 mM Tris, 15 mM (NH₄)₂SO₄, 20% CH3CN in waterpH 7.3 Buffer B: 20% water in CH3CN Flow: 1.5 ml/min Gradient: 1-20 min:10% B to 50% B, 20-22 min: 50% B to 60% B, 22-23 min: 60% B to 10% B,23-30 min 10% B 30-31 min 10% B, flow 0.15 ml/min. Detection: 214 nm

Method 8: HPLC-MS

The following instrumentation is used:

-   -   Hewlett Packard series 1100 G1312A Bin Pump    -   Hewlett Packard series 1100 G13 15A DAD diode array detector    -   Sciex3000 triplequadropole mass spectrometer    -   Gilson 215 micro injector    -   Sedex55 evaporative light scattering detector

Pumps and detectors are controlled by MassChrom 1.1.1 software runningon a Macintosh G3 computer. Gilson Unipoint Version 1.90 controls theauto-injector.

The HPLC pump is connected to two eluent reservoirs containing:

A: 0.01% TFA in water

B: 0.01% TFA in acetonitrile

The analysis is performed at room temperature by injecting anappropriate volume of the sample (preferably 10 μl) onto the column,which is eluted, with a gradient of acetonitrile. The eluate from thecolumn passed through the UV detector to meet a flow splitter, whichpassed approximately 30 μl/min (1/50) through to the API Turbo ion-sprayinterface of API 3000 spectrometer. The remaining 1.48 ml/min (49/50) ispassed through to the ELS detector.

The HPLC conditions, detector settings and mass spectrometer settingsused are giving in the following table.

Column Waters X-Terra C18, 5μ, 50 mm × 3 mm id Gradient 5%-90%acetonitrile linearly during 7.5 min at 1.5 ml/min Detection 210 nm(analogue output from DAD) MS ionisation mode API Turbo ion-spray ELSGain 8 and 40° C.

Method 9: HPLC-MS

-   -   Hewlett Packard series 1100 G1312A Bin Pump    -   Hewlett Packard series 1100 Column compartment    -   Hewlett Packard series 1100 G1315A DAD diode array detector    -   Hewlett Packard series 1100 MSD    -   Sedere 75 Evaporative Light Scattering detector

The instrument was controlled by HP Chemstation software.

The HPLC pump was connected to two eluent reservoirs containing:

A: 0.01% TFA in water

B: 0.01% TFA in acetonitrile

The analysis is performed at 40° C. by injecting an appropriate volumeof the sample (preferably 1 μl) onto the column which is eluted with agradient of acetonitrile.

The HPLC conditions, detector settings and mass spectrometer settingsused are giving in the following:

Column: Waters Xterra MS C-18 × 3 mm id 5 μm Gradient: 5%-100%acetonitrile linear during 7.5 min at 1.5 ml/min Detection: 210 nm(analogue output from DAD) ELS (analogue output from ELS)

After the DAD the flow was divided yielding approx 1 ml/min to the ELSand 0.5 ml/min to the MS.

MALDI-TOF-MS spectra were recorded on a Bruker Autoflex II TOF/TOFoperating in linear mode using a matrix of sinnapinic acid, a nitrogenlaser and positive ion detection. Accelerating voltage: 20 kV.

Example 1 Synthesis ofN^(εB29)-(3-[2-{2-(2-[ω-carboxy-pentadecanoyl-γ-dlutamyl-(2-amino-ethoxy)]-ethoxy)-ethoxy}-ethoxy]-propinoyl)DesB30 Human Insulin

DesB30 human insulin (400 mg, 0.070 mmol) was dissolved in 100 mM Na₂CO₃(5 ml, pH 10.2) at room temperature. Succinimidyl3-[2-{2-(2-[ω-tert-butyl-carboxy-pentadecanoyl-γ-glutamyl-α-tert-butyl-(2-amino-ethoxy)]-ethoxy)-ethoxy}-ethoxy]-propinoyl(72 mg, 0.084 mmol, prepared as described below), was dissolved inacetonitrile (5 ml) and subsequently added to the insulin solution.After 30 mins, 0.2 M methylamine (0.5 ml) was added. pH was adjusted byHCl to 5.5, and the isoelectric precipitate was collected bycentrifugation and dried in vacuo to give 345 mg. The coupling yield was64% (RP-HPLC, C4 column; Buffer A: 10% MeCN in 0.1% TFA-water, Buffer B:80% MeCN in 0.1% TFA-water; gradient 20% to 90% B in 16 minutes). Theprotected product was dissolved in TFA (10 ml), left 30 mins, andevaporated in vacuo. The crude product was dissolved in water andlyophilized.

N^(εB29)-(3-[2-{2-(2-[ω-carboxy-pentadecanoyl-γ-glutamyl-(2-amino-ethoxy)]-ethoxy)-ethoxy}-ethoxy]-propinoyl)desB30 human was purified by RP-HPLC on C4-column, buffer A: 20%EtOH+0.1% TFA, buffer B: 80% EtOH+0.1% TFA; gradient 15-60% B, followedby HPLC on C4-column, buffer A: 10 mM Tris+15 mM ammonium sulphate in20% EtOH, pH 7.3, buffer B: 80% EtOH, gradient 15-60% B. The collectedfractions were desalted on Sep-Pak with 70% acetonitrile+0.1% TFA,neutralized by addition of ammonia and freeze-dried. The unoptimizedyield was 60 mg, 13%. The purity as evaluated by HPLC was >98%.MALDI-TOF-MS 6349, C₂₈₅H₄₃₂N₆₆O₈₆S₆ requires 6351.

Preparation of succinmidyl3-[2-{2-(2-[ω-tert-butyl-carboxy-pentadecanoyl-γ-glytamyl-α-tert-butyl-(2-amino-ethoxy)]-ethoxy)-ethoxy}-ethoxy]-propinoyl).

Hexadecadioic acid (40.0 g, 140 mmol) was suspended in toluene (250 ml)and the mixture was heated to reflux. N,N-dimethylformamidedi-tert-butyl acetal (76.3 g, 375 mmol) was added drop-wise over 4hours. The mixture was refluxed overnight. The solvent was removed invacuo at 50° C., and the crude material was suspended in DCM/AcOEt (500ml, 1:1) and stirred for 15 mins. The solids were collected byfiltration and triturated with DCM (200 ml). The filtrated wereevaporated in vacuo to give crude mono-tert-butyl hexadecandioate, 30grams. This material was suspended in DCM (50 ml), cooled with ice for10 mins, and filtered. The solvent was removed in vacuo to leave 25 gramcrude mono-tert-butyl hexadecandioate, which was recrystallized fromheptane (200 ml) to give mono-tert-butyl hexadecandioate, 15.9 g (33%).Alternatively to recrystallization, the mono-ester can be purified bysilica chromatography in AcOEt/heptane.

¹H-NMR (CDCl₃) δ: 2.35 (t, 2H), 2.20 (t, 2H), 1.65-1.55 (m, 4H), 1.44(s, 9H), 1.34-1.20 (m, 20H).

The mono tert-butyl ester (2 g, 5.8 mmol) was dissolved in THF (20 ml)and treated with TSTU (2.1 g, 7.0 mmol) and DIEA (1.2 ml, 7.0 mmol) andstirred overnight. The mixture was filtered, and the filtrate wasevaporated in vacuo. The residue was dissolved in AcOEt and washed twicewith cold 0.1 M HCl and water. Drying over MgSO4 and evaporation invacuo gave succinimidyl tert-butyl hexadecandioate, 2.02 g (79%).

1H-NMR (CDCl3) δ: 2.84 (s, 4H), 2.60 (t, 2H), 2.20 (t, 2H), 1.74 (p,2H), 1.56 (m, 2H), 1.44 (s, 9H), 1.40 (m, 2H), 1.30-1.20 (m, 18H).

Succinimidyl tert-butyl hexadecandioate (1 g, 2.27 mmol) was dissolvedin DMF (15 ml) and treated with L-Glu-OtBu (0.51 g, 2.5 mmol) and DIEA(0.58 ml, 3.41 mmol) and the mixture was stirred overnight. The solventwas evaporated in vacuo, and the crude product was dissolved in AcOEt,and washed twice with 0.2M HCl, with water and brine. Drying over MgSO₄and evaporation in vacuo gave w-tert-butylcarboxy-pentadecanoyl-L-glutamyl-α-tert-butyl ester, 1.2 g (100%).

1H-NMR (CDCl3) δ: 6.25 (d, 1H), 4.53 (m, 1H), 2.42 (m, 2H), 2.21 (m,4H), 1.92 (m, 1H), 1.58 (m, 4H), 1.47 (s, 9H), 1.43 (s, 9H), 1.43-1.22(m, 18H).

15-tert-butyl-carboxy-pentadecanyl-L-glutamyl-α-tert-butyl ester (1.2 g,2.27 mmol) was dissolved in THF (15 ml) and treated with TSTU (0.82 g,2.72 mmol) and DIEA (0.47 ml, 2.72 mmol) and stirred overnight. Themixture was filtered, and the filtrate was evaporated in vacuo. Theresidue was dissolved in AcOEt and washed twice with cold 0.1 M HCl andwater. Drying over MgSO₄ and evaporation in vacuo gave succinimidylw-tert-butyl-carboxy-pentadecanyl-L-glumtayl-α-tert-butyl ester, 1.30 g(92%).

¹H-NMR (CDCl₃) δ: 6.17 (d, 1H), 4.60 (m, 1H), 2.84 (s, 4H), 2.72 (m,1H), 2.64 (m, 1H), 2.32 (m, 1H), 2.20 (m, 4H), 2.08 (m, 1H), 1.6 (m,4H), 1.47 (s, 9H), 1.43 (s, 9H), 1.33-1.21 (m, 20H).

Succinimidyl 15-tert-butyl-carboxy-pentadecanyl-L-glumtayl-α-tert-butylester (109 mg, 0.17 mmol) was dissolved in DCM (2 ml) and treated with3-(2-{2-[2-(2-amino-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionic acid (51mg, 0.19 mmol, Quanta Biodesign, OH, USA) and DIEA (45 μL, 0.26 mmol).The mixture was stirred overnight and evaporated in vacuo. The residuewas dissolved in AcOEt and washed twice with cold 0.2 M HCl, water andbrine. Drying over MgSO₄ and evaporation in vacuo gave3-[2-{2-(2-[ω-tert-butyl-carboxy-pentadecanoyl-γ-glumtayl-α-tert-butyl-(2-amino-ethoxy)]-ethoxy)-ethoxy}-ethoxy]-propionicacid), 119 mg (88%).

¹H-NMR (CDCl₃) δ: 7.01 (t, 1H), 6.58 (d, 1H), 4.42 (m, 1H), 3.76 (d,2H), 3.62 (m, 16H), 3.55 (t, 2H), 3.42 (m, 1H), 2.58 (t, 2H), 2.28 (m,2H), 2.17 (m, 2H), 2.11 (m, 1H), 1.94 (m, 1H), 1.57 (m, 4H), 1.43 (s,9H), 1.42 (s, 9H), 1.22 (m, 20H).

3-[2-{2-(2-[ω-tert-Butyl-carboxy-pentadecanoyl-γ-glumtayl-α-tert-butyl-(2-amino-ethoxy)]-ethoxy)-ethoxy}-ethoxy]-propionicacid) (119 mg, 0.15 mmol) was dissolved in THF (2 ml) and treated withTSTU (55 mg, 018 mmol) and DIEA (31 μL, 0.18 mmol) and stirredovernight. The mixture was filtered, and the filtrate was evaporated invacuo. The residue was dissolved in AcOEt and washed twice with cold 0.1M HCl and water. Drying over MgSO₄ and evaporation in vacuo gavesuccinimidyl3-[2-{2-(2-[ω-tert-butyl-carboxy-pentadecanoyl-γ-glutamyl-α-tert-butyl-(2-amino-ethoxy)]-ethoxy)-ethoxy}-ethoxy]-propinoyl),123 mg (92%).

¹H-NMR (CDCl₃) δ: 6.64 (t, 1H), 6.54 (d, 1H), 4.35 (m, 1H), 3.80 (d,2H), 3.59 (m, 16H), 3.51 (t, 2H), 3.39 (m, 1H), 2.85 (t, 2H), 2.79 (s,4H), 2.22 (m, 2H), 2.15 (m, 2H), 2.08 (m, 1H), 1.90 (m, 1H), 1.55 (m,4H), 1.41 (s, 9H), 1.39 (s, 9H), 1.20 (m, 20H).

Example 2 Synthesis ofN^(εB29)-(3-[2-{2-(2-[ω-carboxy-hebtadecanoyl-γ-glutamyl-(2-amino-ethoxy)]-ethoxy)-ethoxy}-ethoxy]-propinoyl)DesB30 Human Insulin

This compound was prepared in analogy with example 1 via reaction ofL-GluOtBu with tert-butyl succinimidyl octadecandioate followed byactivation with TSTU, activation with TSTU, reaction with3-(2-{2-[2-(2-Amino-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionic acidactivation with TSTU, coupling with DesB30 human insulin anddeprotection by TFA.

MALDI-TOF-MS 6380, calculated 6379.

Example 3 Synthesis ofN^(εB29)-{3-[2-(2-{2-[2-(15-carboxy-pentadecanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionyl-γ-glutamylDesB30 Human Insulin

Step 1:ω-[2-(2-{2-[2-(2-Carboxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethylcarbamoyl]-pentadecanoicacid tert-butyl ester

Hexadecanedioic acid tert-butyl ester 2,5-dioxo pyrrolidin-1-yl ester(0.12 g, 0.283 mmol) was dissolved in DMF (2.5 ml),3-(2-{2-[2-(2-amino-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionic acid (75mg, 0.283 mmol) was added and the mixture was stirred at rt for 16 h.The reaction mixture was combined with another reaction mixtureperformed on a 0.038 mmol scale. AcOEt (25 ml) was added an the solutionwas washed with acidified water (15 ml+300 μl of 0.1 N HCl) and water(3×15 ml), dried over MgSO₄ and concentrated under vacuum, adding someDCM and concentrating again twice, thus yielding a white greasy residue(0.15 g, 79%)

HPLC-MS m/z: 590 (M+1), Rt=5.24 min.

1H-NMR (CDCl₃, 400 MHz) δ 6.48 (br, 1H), 3.79 (t, 2H), 3.6-3.7 (m, 14H),3.47 (m, 2H), 2.60 (t, 2H), 2.17-2.22 (m, 4H), 1.57-1.64 (m, 4H), 1.44(s, 9H), 1.2-1.3 (m, 20H).

Step 2:2-{3-[2-(2-{2-[2-(ω-tert-Butoxycarbonylpentadecanoylamino)ethoxy]-ethoxy}ethoxy)ethoxy]propionylamino}pentanedioicacid 5-benzyl ester 1-tert-butyl ester

Hexadecanoic acid tert-butyl ester 0.15 g, 0.254 mmol) was dissolved inDMF (2.5 ml) and HOBt (48 mg, 0.356 mmol) and EDAC (63 mg, 0.331 mmol)were added. The solution was stirred at it for 30 min andH-Glu-(OBzl)-OtBu (117 mg, 0.356 mmol) was added. The reaction wasstirred at it for 16 h, and AcOEt (25 ml) was added. The solution waswashed with water (10 ml), 0.2 N HCl (3×10 ml), 1:1 Sat. NaCl/water(3×10 ml), dried over MgSO₄ and concentrated to yield an oil (0.24 g).The product was purified by flash chrometography (silica, 95:5DCM/methanol) to yield an oil 0.2g.

HPLC-MS (method 9): m/z: 866 (M+1), R_(t)=6.99-7.09 min

1H-NMR (CDCl₃, 400 MHz) δ 7.34-7.38 (m, 5H), 6.83 (d, 1H), 6.10 (br,1H), 5.11 (s, 2H), 4.50-4.55 (m, 1H), 3.71-3.75 (m, 2H), 3.60-3.65 (m,12H), 3.55 (t, 2H), 3.36-3.42 (m, 2H), 2.36-2.51 (m, 4H), 2.14-2.24 (m,5H), 1.93-2.00 (m, 1H), 1.57-1.63 (m, 4H), 1.46 (s, 9H), 1.44 (s, 9H),1.2-1.3 (m, 20H).

Step 3:2-{3-[2-(2-{2-[2-(ω-tert-butoxycarbonyl-pentadecanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]-propionylamino}pentanedioicacid 1-tert-butyl ester

2-{3-[2-(2-{2-[2-(ω-tert-Butoxycarbonylpentadecanoylamino)ethoxy]-ethoxy}ethoxy)ethoxy]propionylamino}pentanedioicacid 5-benzyl ester 1-tert-butyl ester (0.2 g, 0.23 mmol) was dissolvedin THF. The flask was filled with N₂, and palladium (0.3 g, 10% oncarbon, 50% water) was added, and the flask was equipped with a balloonfilled with H₂. The mixture was stirred for 16 h at rt, and filteredthrough celite, washing with THF. The filtrate was concentrated to yieldan oil (0.16 g, 89%).

HPLC-MS (method 9m/z: 775 (M+1), Rt=5.46 min.

Step 4:2-{3-[2-(2-{2-[2-(ω-tert-Butoxycarbonyl-pentadecanoylamino)ethoxy]-ethoxy}ethoxy)ethoxy]-propionylamino}pentanedioicacid 5-tert-butyl ester 1-(2,5-dioxopyrrolidin-1-yl)ester

2-{3-[2-(2-{2-[2-(ω-tert-Butoxycarbonyl-pentadecanoylamino)ethoxy]-ethoxy}ethoxy)ethoxy]-propionylamino}pentanedioicacid 1-tert-butyl ester (0.16 g, 0.21 mmol) was dissolved in DMF (2 ml)and THF (4 ml) and DIEA (42 pl, 0.25 mmol) was added. The solution wascooled to 0° C., and TSTU (74 mg, 0.21 mmol) was added. The reaction wasstirred over night at rt. the solvent was removed under vacuum and AcOEt(25 ml) was added. The mixture was washed with 0.2 N HCl (3×10 ml), satNaHCO₃ (3×10 ml), dried over MgSO₄ and concentrated under vacuum toyield an oil (0.16 g). The product was purified by flash chromatography(silica, 95:5 DCM/methanol) to yield an oil (0.11 g, 61%).

HPLC-MS (method 9) m/z: 872 (M+1), Rt=5.67 min.

Step 5:N^(εB29)-{3-[2-(2-{2-[2-(ω-carboxy-pentadecanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionyl-δ-glutamylDesB30 Human Insulin

2-{3-[2-(2-{2-[2-(ω-tert-Butoxycarbonyl-pentadecanoylamino)ethoxy]-ethoxy}ethoxy)ethoxy]-propionylamino}pentanedioicacid α-tert-butyl ester 1-(2,5-dioxopyrrolidin-1-yl)ester was coupled todesB30 insulin in similar fashion as described in Example 1 Theintermediate product was purified by preparative HPLC (C₁₈-5 cm dia.)before treating with TFA. The final product was purified by preparativeHPLC (C₄, 2 cm dia.) then (C₄, 1 cm dia.) (20-60% acetonitrile).

MALDI-TOF-MS: 6355, Calculated: 6351

Example 4 Synthesis ofN^(εB29)-(ω-[2-(2-{2-[2-(2-carboxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethylcarbamoyl]-hebtadecanoyl-α-glutamyl)DesB30 Human Insulin

This compound was prepared in analogy with example 1 via reaction ofH₂N(CH₂CH₂O)₄CH₂CH₂COOtBu (Quanta Biodesign, OH, USA) withmono-succinimidyl oc-tadecandioate followed by activation with TSTU,reaction with L-Glu(OtBu), activation with TSTU, coupling with DesB30human insulin and deprotection by TFA. LCMS 6380, method 6, calculated6379.

Example 5 Synthesis ofN^(εB29)-(ω-[2-(2-{2-[2-(2-carboxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethylcarbamoyl]-heptadecanoyl-γ-glutamyl)DesB30 Human Insulin

This compound was prepared in analogy with example 1 via reaction ofH₂N(CH₂CH₂O)₄CH₂CH₂COOtBu (Quanta Biodesign, OH, USA) withmono-succinimidyl oc-tadecandioate followed by activation with TSTU,reaction with L-Glu-OtBu, activation with TSTU, coupling with DesB30human insulin and deprotection by TFA.

LCMS 6378.4, method 6, calculated 6379.4.

Example 6 Synthesis ofN^(εB29)-3-[2-(2-{2-[2-(ω-carboxy-heptadecanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionyl-γ-glutamylDesB30 Human Insulin

The compound was prepared in the same manner as withN^(εB29)-3-[2-(2-{2-[2-(ω-carboxy-pentadecanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionyl-γ-glutamyldesB30 insulin using octadecanedioic acid tert-butyl ester2,5-dioxo-pyrrolidin-1-yl ester as the starting material.

Step 1:ω-[2-(2-{2-[2-(2-carboxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethylcarbamoyl]-heptadecanoicacid tert-butyl ester

HPLC-MS (method 9) m/z: 618 (M+1), Rt=5.92 min.

1H-NMR (CDCl₃, 300 MHz) δ 6.46 (br, 1H), 3.79 (t, 2H), 3.61-3.69 (m,14H), 3.44-3.49 (m, 2H), 2.60 (t, 2H), 2.16-2.22 (m, 4H), 1.51-1.68 (m,4H), 1.44 (s, 9H), 1.19-1.36 (m, 24H).

Step 2:2-{3-[2-(2-{2-[2-(17-tert-Butoxycarbonylheptadecanoylamino)ethoxy]-ethoxy}ethoxy)ethoxy]-propionylamino}pentanedioicacid 5-benzyl ester 1-tert-butyl ester

HPLC-MS (method 9) m/z: 894 (M+1), Rt=7.82-7.89 min. 1H-NMR (CDCl₃, 300MHz) 8 7.29-7.42 (m, 5H), 6.83 (d, 1H), 6.13 (br, 1H), 5.11 (s, 2H),4.46-4.59 (m, 1H), 3.68-3.81 (m, 2H), 3.57-3.68 (m, 12H), 3.55 (t, 2H),3.39-3.49 (m, 2H), 2.32-2.55 (m, 4H), 2.12-2.28 (m, 5H), 1.86-2.07 (m,1H), 1.51-1.68 (m, 4H), 1.46 (s, 9H), 1.44 (s, 9H), 1.17-1.36 (m, 24H).

Step 3:2-{3-[2-(2-{2-[2-(17-tert-Butoxycarbonylheptadecanoylamino)ethoxy]-ethoxy}ethoxy)ethoxy]propionylamino}pentanedioicacid 1-tert-butyl ester

HPLC-MS (method 9) m/z: 804 (M+1), Rt=5.81 min.

Step 4:2-{3-[2-(2-{2-[2-(ω-tert-Butoxycarbonyl-heptadecanoylamino)ethoxy]-ethoxy}ethoxy)ethoxy]propionylamino}pentanedioicacid 5-tert-butyl ester 1-(2,5-dioxo-pyrrolidin-1-yl)ester

HPLC-MS (method 9) m/z: 901 (M+1), Rt=6.00 min.

1H-NMR (CDCl₃, 300 MHz) δ 6.94 (d, 1H), 6.15 (br, 1H), 4.55-4.62 (m,1H), 3.71-3.79 (m, 2H), 3.59-3.71 (m, 12H), 3.55 (t, 2H), 3.42-3.47 (m,2H), 2.84 (s, 4H), 2.58-2.79 (m, 2H), 2.52 (t, 2H), 2.24-2.41 (m, 1H),2.13-2.24 (m, 4H), 2.04-2.10 (m, 1H), 1.51-1.70 (m, 4H), 1.48 (s, 9H),1.44 (s, 9H) 1.19-1.37 (m, 24H).

Step 5:N^(εB29)-(3-[2-(2-{2-[2-(17-carboxy-heptadecanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionylgamma-glutamyl DesB30 Human Insulin

The final product was purified by HPLC (C₁₈-5cm dia.).

HPLC-MS (method 9) m/z: 1596.4 (M+4/4), Calculated 6379, Rt=4.05 min

Example 7 Synthesis ofN^(εB29)-(3-(3-{2-[2-(3-17-carboxyheptanoylamino]propoxy)ethoxy]-ethoxy}propylcarbamoyl)propionyl)DesB30 Human Insulin

This compound was prepared using the same synthesis steps as reportedfor the synthesis of example 1.

Step 1.N-(3-{2-[2-(3-tert-Butoxycarbonylaminopropoxy)ethoxy]ethoxy}propyl)succinamicacid

preparation from1-(tert-butoxycarbonylamino)-4,7,10-trioxa-13-tridecanamine (5g) andsuccinic anhydride (1.98) gave 7 g crude product. LCMS (Method 6): Rt3.34 min; m/z (M+1) 421 Calcd: 421

Step 2.7-[3-(2-{2-[3-(3-Carboxypropionylamino)-propoxy]-ethoxy}-ethoxy)-propylcarbamoyl]-heptanoicacid tert-butyl ester

This compound was prepared by deprotection ofN-(3-{2-[2-(3-tert-butoxycarbonylaminopropoxy)-ethoxy]ethoxy}propyl)succinamicacid (1.56 mmol) by means of TFA, followed by reaction with octanedioicacid tert-butyl ester 2,5-dioxo-pyrrolidin-1-yl ester (1.56 mmol). asdescribed in example 8 step3.

The crude product was purified on Gilson using acidic HPLC on a C18column (Jones, Kromasil RP18 5 μm 15×225 mm).

Gradient: 0.0-4.0 min 20% A; 4.0-11.0 min 20-90% A; 11-16 min 90% A.

The product was collected in fractions from 15.0-17.0 min. The combinedfractions were evaporatied yielding the wanted product (0.78 g)

LCMS (Method 9): Rt 4.03 min; m/z (M+1) 533, Calcd.: 533.

Step 3.7-{3-[2-(2-{3-[3-(2,5-Dioxopyrrolidin-1-yloxycarbonyl)-propionylamino]-propoxy}-ethoxy)-ethoxy]-propylcarbamoyl}-heptanoicacid tert-butyl ester

7-[3-(2-{2-[3-(3-Carboxypropionylamino)-propoxy]-ethoxy}-ethoxy)-propylcarbamoyl]-heptanoicacid tert-butyl ester (0.78g,1.46 mmol) was activated by means of TSTUas described in example 8 step 4. Crude yield 360 mg, LCMS Method 6: Rt4.40 min; m/z (M+1) 630; Calcd.: 630. The compound was used withoutfurther purification.

Step 4.N^(εB29)-(3-(3-{2-[2-(3-[7-carboxyheptanoylamino]propoxy)ethoxy]-ethoxy}propylcarbamoyl)propionyl-γ-glutamyl)DesB30 Human Insulin

Preparation following step 6 in example 8 resulted in 0.78 g of thetarget product after purification on Gilson using acidic HPLC on a C18column (Jones, Kromasil RP18 5 μm 15×225 mm). Gradient: 0.0-1.0 min: 30%CH3CN, 1.00-15.0 min: 30-50% CH3CN, 15.0-20.0 min: 50% CH3CN Flow: 10ml/min. Rt=14.5-16.0 min.

MALDI-TOF-MS (matrix SA): m/z 6167; calc. 6165.

HPLC (method 5); Rt 3.973 min.

Example 8 Synthesis ofN^(εB29)-(3-(3-{4-[3-(7-Carboxyheptanoylamino)propoxy]butoxy}propylcarbamoyl)-propionyl-γ-glutamyl)DesB30 Human Insulin

Step 1:N-{3-[4-(3-tert-Butoxycarbonylaminopropoxy)-butoxy]-propyl}succinamicacid

1-(tert-Butoxycarbonylamino)-4,9-dioxa-12-dodecanamine (5.0 g, 16.45mmol) was dissolved in THF (30 mL), succinic anhydride (1.81 g, 18.1mmol) in acetonitrile (10 mL) was added and the mixture was heated to 60C for 4 h, and subsequently stirred at RT over-night.

The mixture was evaporated to dryness and EtAc (50 mL) was added.

The EtAc phase was washed with HCl (0.1 M) 3 times, dried with MgSO₄ andsubsequently the organic phase was evaporated to dryness which gave 5.86g (88%) of a thick oil.

LCMS (Method 6): Rt 2.86 min; m/z (M+1) 405. Calcd: 405.

This product was used without further purification.

Step 2. Octanedioic acid tert-butyl ester 2,5-dioxo-pyrrolidin-1-ylester

Octanedioic acid mono-tert-butyl ester (3.14 g, 13.63 mmol) wasdissolved in THF (100 mL). TSTU (4.9 g, 16.3 mmol) was added and pH wasadjusted to 8.5 with DIPEA (2.85 mL).

The mixture was stirred under nitrogen overnight, evaporated to dryness,dissolved in EtAc (50 mL) which subsequently was extracted 2 times withHCL (0.1 M). The organic phase was dried with MgSO₄, filtered andevaporated resulting in an slightly yellow oil (5 g, containing smallamounts of solvent)

LCMS (Method 6): Rt 6.56 min; m/z (M+1) 328. Calcd: 328.

Step 3:7-(3-{4-[3-(3-Carboxypropionylamino)propoxy]butoxy}propylcarbamoyl)heptanoicacid tert-butyl ester

N-{3-[4-(3-tert-butoxycarbonylaminopropoxy)-butoxy]-propyl}succinamicacid (4.60 g, 11.37 mmol) was stirred with TFA (20 mL) at RT for 60 min,after evaporation the residue was stripped with DCM (30 mL×2) andevaporated to dryness.

The resulting oil was dissolved in acetonitrile (30 mL) and octanedioicacid tert-butyl ester 2,5-dioxo-pyrrolidin-1-yl ester (4.46 g, 13.6mmol) in DMF (20 mL) was added.

pH was adjusted to 8.5 with DIPEA and the mixture was stirred overnightunder nitrogen. The mixture was subsequently evaporated to dryness andredissolved in EtAc (50 mL). The EtAc phase was extracted ×3 with HCl(0.1 M), the organic layer dried over magnesium sulphate, filtered andevaporated resulting in a slightly yellow crystalline oil (6.5 g,content of solvent residues)

LCMS (Method 6): Rt 4.31 min; m/z (M+1) 517. Calcd: 517.

The crude product was used for further reaction without furtherpurification.

Step 4.2-[3-(3-{4-[3-(7-tert-butoxycarbonylheptanoylamino)propoxy]butoxy}-propylcarbamoyl)-propionylamino]pentanedioicacid 1-tert-butyl ester

7-(3-{4-[3-(3-Carboxypropionylamino)propoxy]butoxy}propylcarbamoyl)heptanoicacid tert-butyl ester (5.9 g), the crude product from above, wasdissolved in THF (20 mL), TSTU (4.13 g, 13.7 mmol) was added togetherwith DMF (6 mL), pH was adjusted to 8.2 with DIPEA (2.6 mL). The mixturewas stirred overnight under nitrogen.

The mixture was evaporated and the residue dissolved in EtAc which wasextracted with HCl (0.1 M) 3 times.

The organic layer was dried with magnesium sulphate, filtered and thefiltrate evaporated to give an oil.

LCMS (Method 6): Rt 4.57 min; m/z 614 corresponding to the activatedacid.

This was dissolved in THF (30 mL), pH was adjusted to 8.2 with DIPEA(0.4 mL) and H-glu-OtBu (1.7 g, 4.9 mmol) was added together with DMF(10 mL). The mixture was stirred at RT for 3 h, filtration followed byevaporation afforded a thick yellow oil.

This was extracted between EtAc and HCl (0.1 M) as reported above, andthe resulting dried EtAc layer gave 3.5 g crude product on evaporation.LCMS (Method 6): Rt 4.77 min; m/z (M+1) 702.

The crude product was purified on Gilson using acidic HPLC on a C18column (Jones, Kromasil RP18 5 μm 15×225 mm).

Gradient: 0.0-10.0 min 35% A; 10.0-25.0 min 35-80% A; 25-30 min 90% A;30-35 min 100% A.

The product was collected in fractions from 21-22.5 min. The combinedfractions were evaporated yielding the wanted product (1.8 g)

LCMS (Method 6): Rt 4.77 min; m/z (M+1) 702, Calcd. 702.

Step 5.2-[3-(3-{4-[3-(7-tert-butoxycarbonylheptanoylamino)propoxy]butoxy}propylcarbamoyl)-propionylamino]pentanedioicacid 5-tert-butyl ester 1-(2,5-dioxopyrrolidin-1-yl)ester

2-[3-(3-{4-[3-(7-tert-butoxycarbonylheptanoylamino)propoxy]butoxy}-propylcarbamoyl)-propionylamino]pentanedioicacid 1-tert-butyl ester (1.5 g, 2.14 mmol) was dissolved in THF (20 mL),pH was adjusted to 8.5 with DIPEA (0.9 mL), TSTU (0.83 g, 2.77 mmol) wasadded in DMF (5 mL). The mixture was stirred under nitrogen overnight ,subsequent evaporation and extraction between EtAc and HCl as describedabove resulted in 1.75 g crude product.

LCMS (Method 6): Rt 5.10 min; m/z (M+1) 800, Calcd.: 800.

Step 6.N^(εB29)-(3-(3-{4-[3-(7-carboxyheptanoylamino)propoxy]butoxy}propylcarbamoyl)-propionyl-δ-glytamyl)DesB30 Human Insulin

2-[3-(3-{4-[3-(7-tert-butoxycarbonylheptanoylamino)propoxy]butoxy}propylcarbamoyl)-propionylamino]pentanedioicacid 5-tert-butyl ester 1-(2,5-dioxopyrrolidin-1-yl)ester (0.255 g,0.319 mmol) was dissolved in acetonitrile (10 mL) and added to asolution of desB30 human insulin (1.82 g) dissolved in Na₂CO₃ solution(10 mL, pH 10.3), pH was adjusted to 10.1 with NaOH (0.1 M). The mixturewas stirred at RT for 2 h, then pH was adjusted to 5.5 by means of HCl(2M, 3 mL) resulting in the precipitation of an oily crystalline mass.

This was isolated and dissolved in water acetic acid (1M) and freezedried.

The resulting product was dissolved in water and purified on Gilsonusing acidic HPLC on a C18 column (Jones, Kromasil RP18 5 μm 15×225 mm).

Gradient: 0.0-5.0 min 35% A; 5.0-25.0 min 35-80% A; 25-30 min 90% A;30-35 min 100% A. Fractions around Rt 15 min were collected, mixed andevaporated.

The product was treated with TFA/DCM 1/1 (20 mL) by stirring at RT for 1h, subsequent evaporation to dryness and stripping with DCM 40 mL×2resulted in the deprotected product which was dissolved in water andfreeze dried giving 540 mg of the wanted product.

MALDI.TOF-MS: m/z 6276.66; calc. 6276.

HPLC (method 5); Rt 10.19 min.

Example 9 Synthesis ofN^(εB29)-(3-(3-{2-[2-(3-[9-Carboxynonanoylamino]propoxy)ethoxy]ethoxy}-propylcarbamoyl)propionyl)DesB30 Human Insulin

Following the procedure from example 7, but exchanging the diacid partgave the product.

Preparation following step 6 in example 8 using 0.114 mmol of desB30insulin resulted in 0.96 g of the protected compound.

Gilson purification using acidic HPLC on a C18 column (Jones, KromasilRP18 5 μm 15×225 mm). Gradient: 0.0-1.0 min: 35% CH3CN, 1.00-15.0 min:35-55% CH3CN, 15.0-20.0 min: 55% CH3CN Flow: 10 ml/min. Rt=12.5-14.0min.

Deprotection my means of TFA gave 0.141 g colourless compound afterfreeze drying.

MALDI-TOF-MS (matrix SA): m/z 6195; calc. 6186

HPLC (method 5): Rt; 4.094 min.

Decanedioic acid tert-butyl ester 2,5-dioxo-pyrrolidin-1-yl ester

Preparation as described in step 2 example 8 gave 5.57 g crude productwhich was used without further purification. LCMS (Method 6): Rt 5.82min; m/z (M+1) 356, Calcd.: 355.

Example 10 Synthesis ofN^(εB29)-(3-(2-{2-[2-(9-carboxynonanoylamino)ethoxy]ethoxy}ethylcarbamoyl)propionyl-γ-olutamyl)DesB30 Human Insulin

The preparation was performed using the methodology described in example8

(S)-2-[3-(2-{2-[2-(9-tert-Butoxycarbonylnonanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionylamino]pentanedioicacid 1-tert-butyl ester (0.59 g, 0.876 mmol) was activated with TSTU,0.132 g (0.171 mmol) the crude reaction product was reacted with desB30insulin (0.154 mmol) as described in example 8

This resulted in 740 mg of oily precipitate which was freeze-dried andpurified on

Gilson using acidic HPLC on a C18 column (Jones, Kromasil RP18 5 μm15×225 mm).

Gradient: 0.0-5.0 min 30% A; 5.0-20.0 min 35-50% A.

105 mg of target compound was isolated. MALDI.TOF-MS (matrix Cyano): m/z6245.9; calc. 6243.

HPLC (method 5); Rt 8.759 min.

N-{2-[2-(2-tert-butoxycarbonylamino-ethoxy)-ethoxy]-ethyl}-succinamicacid

Preparation from 1-(t-butyloxycarbonylamino)-3,6-dioxa-8-octaneamine) (5g, 20.16 mmol) and succinic anhydride (2.218 g, 22,18 mmol) gave a thickyellow oil which crystallised on standing (6.5 g, yield 98%). LCMS(Method 6): Rt 2.99 min; m/z (M+1) 349; Calcd.: 349.

9-(2-{2-[2-(3-carboxypropionylamino)ethoxy]ethoxy}ethylcarbamoyl)nonanoicacid tert-butyl ester

Preparation from decanedioic acid tert-butyl ester2,5-dioxo-pyrrolidin-1-yl ester (1.13 g, 3.45 mmol) andN-{2-[2-(2-tert-butoxycarbonylamino-ethoxy)-ethoxy]-ethyl}-succinamicacid (1 g, 2.84 mmol) as described in step 3 example 8 gave 1.68 g crudeproduct which was used without further purification. LCMS (Method 6): Rt3.86 min; m/z (M+1) 489; Calcd.: 489.

(S)-2-[3-(2-{2-[2-(9-tert-Butoxycarbonylnonanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionylamino]pentanedioicacid 1-tert-butyl ester

Preparation from9-(2-{2-[2-(3-carboxypropionylamino)ethoxy]ethoxy}-ethylcarbamoyl)nonanoicacid tert-butyl ester (1.4 g, 2.86 mmol) and glu-OtBu (0.87 g, 4.29mmol) folllowing the method described step4 example 8 gave 1.8 g crudeproduct. LCMS (Method 6): Rt 5.1 min; m/z (M+1) 674; Calcd.: 674.

Gilson purification using acidic HPLC on a C18 column (Jones, KromasilRP18 5 μm 15×225 mm), Gradient: 0.0-10.0 min: 35% CH₃CN, 10.00-25.0 min:35-90% CH3CN, Flow: 10 ml/min. Fractions at Rt=20.0-25.0 min werecollected and evaporated to dryness giving 0.590 g of a yellow oil. LCMS(Method 6): Rt 5.1 min; m/z (M+1) 674; Calcd.: 674.

Example 11 Synthesis ofN^(εB29)-(3-(3-{4-[3-(9-Carboxynonanoylamino)propoxy]butoxy}-propylcarbamoyl)propionyl-γ-glutamyl)DesB30 Human Insulin

(S)-2-[3-(3-{4-[3-(9-tert-Butoxycarbonylnonanoylamino)propoxy]butoxy}-propylcarbamoyl)propionylamino]pentanedioicacid 5-tert-butyl ester 1-(2,5-dioxopyrrolidin-1-yl)ester (0.06 g, 0.073mmol) and desB30 insulin (0.065 mmol) were reacted as described inexample 8.The TFA treated product was purified on Gilson using acidicHPLC on a C18 column (Jones, Kromasil RP18 5 μm 15×225 mm).

Gradient: 0.0-5.0 min 30% A; 5.0-20.0 min 35-50% A. fractions at Rt 16.0min-17.5 min were collected evaporated and subsequently freeze-dried.Yield 34 mg.

MALDI.TOF-MS: m/z 6305.69; calc. 6299.

HPLC method 5; Rt 8.850 min.

9-(3-{4-[3-(3-Carboxypropionylamino)propoxy]butoxy}propylcarbamoyl)nonanoicacid tert-butyl ester

Preparation from decanedioic acid tert-butyl ester2,5-dioxo-pyrrolidin-1-yl ester (0.88 g,

2.47 mmol) andN-{3-[4-(3-tert-butoxycarbonylaminopropoxy)-butoxy]-propyl}succinamicacid

(1 g, 2.47 mmol) as described in example 8 afforded 150 mg compoundafter purification.

LCMS (Method 6): Rt 4.31 min; m/z (M+1) 545; Calcd.: 545.

(S)-2-[3-(3-{4-[3-(9-tert-butoxycarbonylnonanoylamino)propoxy]butoxy}propylcarbamoyl)propionylamino]pentanedioicacid 1-tert-butyl ester

9-(3-{4-[3-(3-Carboxypropionylamino)propoxy]butoxy}propylcarbamoyl)nonanoicacid tert-butyl ester (0.15 g, 0.276 mmol) was activated with TSTU , theresulting OSu-derivative was reacted with H-Glu-OtBu (0.076 g, 0.37mmol) as described previously. After work up the resulting oil waspurified on Gilson using acidic HPLC on a C18 column (Jones, KromasilRP18 5 μm 15×225 mm).

Gradient: 0.0-5.0 min 20% A; 5.0-20.0 min 20-90% A. fractions at Rt 24.5min-25.5 min were collected evaporated and subsequently freeze-dried.Yield 50 mg. LCMS Method 6: Rt 5.43 min; m/z (M+1) 730; Calcd.: 730.

This compound was activated with TSTU resulting in 60 mg crude(S)-2-[3-(3-{4-[3-(9-tert-Butoxycarbonylnonanoylamino)propoxy]butoxy}propylcarbamoyl)propionylamino]-pentanedioicacid 5-tert-butyl ester 1-(2,5-dioxopyrrolidin-1-yl)ester

LCMS Method 6: Rt 5.79 min; m/z (M+Na) 850; Calcd.: 850.

The crude product was used without further purification.

Example 12 Synthesis ofN^(εB29)-(2-[3-(2-(2-{2-(7-carboxyheptanoylamino)ethoxy}ethoxy)-ethylcarbamoyl]propionyl-γ-glutamyl)DesB30 Human Insulin

(S)-2-[3-(2-{2-[2-(7-tert-Butoxycarbonylheptanoylamino)ethoxy]ethoxy}ethylcarbamoyl)-propionylamino]pentanedioicacid 5-tert-butyl ester 1-(2,5-dioxopyrrolidin-1-yl)ester (0.126 g, 0.17mmol) was reacted with desB30 insulin (0.153 mmol) as described above.The crude product after TFA treatment (0.750 mg) was purified two timeson Gilson using acidic HPLC on a C18 column (Jones, Kromasil RP18 5 μm15×225 mm).

Gradient: 0.0-5.0 min 25% A; 5.0-20.0 min 20-50%. Fractions from Rt21.0-22.0 min collected and evaporated resulting in 13 mg compound.

MALDI.TOF-MS (matrix SA): m/z 6221.15; calc. 6215.

LCMS (Method 6): Rt 3.53 min; m/z (M+414) 1556; Calcd.: 1554

7-(2-{2-[2-(3-Carboxy-propionylamino)-ethoxy]-ethoxy}-ethylcarbamoyl)-heptanoicacid tert-butyl ester

Octanedioic acid tert-butyl ester 2,5-dioxo-pyrrolidin-1-yl ester (1.13g, 3.45 mmol) andN-{2-[2-(2-tert-butoxycarbonylamino-ethoxy)-ethoxy]-ethyl}-succinamicacid (1 g, 2.874 mmol) were reacted as described above. 1.75 g crudeproduct was isolated and used without further purification. LCMS (Method6): Rt 3.86 min; m/z (M+1) 461; Calcd.: 461.

(S)-2-[3-(2-{2-[2-(7-tert-Butoxycarbonylheptanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionylamino]pentanedioicacid 1-tert-butyl ester

7-(2-{2-[2-(3-Carboxy-propionylamino)-ethoxy]-ethoxy}-ethylcarbamoyl)-heptanoicacid tert-butyl ester (1.3 g, 2.83 mmol) was activated with TSTU andsubsequently the crude product was reacted with H-glu-OtBu (0.86 g, 4.2mmol). After work up using the method described in example 8, theproduct was further purified on Gilson using acidic HPLC on a C18 column(Jones, Kromasil RP18 5 μm 15×225 mm).

Gradient: 0.0-10.0 min 30% A; 10.0-25.0 min 30-90% A, fractions at Rt20-25 min were collected and evaporated resulting in 600 mg productwhich was used for TSTU activation described below. LCMS (Method 6): Rt4.51 min; m/z (M+1) 646; Calcd.: 646.

(S)-2-[3-(2-{2-[2-(7-tert-Butoxycarbonylheptanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionylamino]pentanedioicacid 5-tert-butyl ester 1-(2,5-dioxopyrrolidin-1-yl)ester

(S)-2-[3-(2-{2-[2-(7-tert-Butoxycarbonylheptanoylamino)ethoxy]ethoxy}-ethylcarbamoyl)propionylamino]pentanedioicacid 1-tert-butyl ester (0.6 g, 0.93 mmol) was activated with TSTU usingthe procedure described above.

This resulted in 0.75 g crude compound which was used without furtherpurification.

LCMS (Method 6): Rt 4.81 min; m/z (M+1) 743; Calcd.: 743

Example 13 Synthesis ofN^(εB29)-(3-[2-(2-{2-[2-(15-carboxypentadecanoylamino)ethoxy]ethoxy}ethoxy)-ethoxy]propionyl))DesB30 Human Insulin

This compound was prepared similarly as described in example 4. Theintermediate15-[2-(2-{2-[2-(2-Carboxyethoxy)ethoxy]ethoxy}ethoxy)ethylcarbamoyl]pentadecanoicacid tert-butyl ester was activated to the OSu-ester using TSTU andcoupled to desB30 human insulin. Deprotection using TFA afforded thetitle compound.

MALDI-TOF MS: m/z=6222. Calculated: 6222

HPLC (Method 1): R_(t)=11.12 min.

HPLC (Method 5): R_(t)=12.03 min.

Example 14 Synthesis ofN^(εB29)-(3-(2-{2-[2-(2-{2-[2-(2-{2-[2-(2-{2-[2-(13-carboxy-tridecanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionoyl-γ-glutamyl)DesB30 Human Insulin

This compound was prepared in analogy with example 1 via reaction ofH₂N(CH₂CH₂O)₁₂CH₂CH₂COOH (Quanta Biodesign, OH, USA) with tert-butylO-succinimidyl tetradecandioate followed by activation with TSTU,reaction with L-Glu-OtBu, activation with TSTU, coupling with DesB30human insulin and deprotection by TFA.

LCMS 6676.0, method 6, calculated 6675.8.

Example 15 Synthesis ofN^(εB29)-(3-[2-(2-{2-[2-(13-Carboxy-tridecanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionoy-γ-glutamyl)DesB30 Human Insulin

This compound was prepared in analogy with example 1 via reaction ofH₂N(CH₂CH₂O)₄CH₂CH₂COOH (Quanta Biodesign, OH, USA) with tert-butylO-succinimidyl tetradecandioate followed by activation with TSTU,reaction with L-Glu-OtBu, activation with TSTU, coupling with DesB30human insulin and deprotection by TFA.

LCMS 6323.2, (method 6) calculated 6323.3.

Example 16 Synthesis ofN^(εB29)-(3-[2-(2-{2-[2-(2-{2-[2-(13-carboxy-tridecanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-propionoyl-γ-glutamyl)DesB30 Human Insulin

This compound was prepared in analogy with example 1 via reaction ofH₂N(CH₂CH₂O)₈CH₂CH₂COOH (Quanta Biodesign, OH, USA) with tert-butylO-succinimidyl tetradecandioate followed by activation with TSTU,reaction with L-Glu-OtBu, activation with TSTU, coupling with Des(B30)human insulin and deprotection by TFA.

LCMS 6498.8, method 6, calculated 6499.6.

Example 17 Synthesis ofN^(εB29)-(3-(2-{2-[2-(15-Carboxy-pentadecanoylamino)-ethoxy]-ethoxy}-ethylcarbamoyl)-propionyl-γ-glutamyl)DesB30 Human Insulin

MALDI-TOF MS (matrix: SA): m/z=6336. Calculated: 6334

HPLC (Method 1): R_(t)=11.71 min.

HPLC (Method 5): R_(t)=9.37 min.

Example 18 Synthesis ofN^(εB29)-(3-(3-{2-[2-(3-[15-Carboxypentadecanoylamino]propoxy)ethoxy]-ethoxy}propylcarbamoyl)propionyl-γ-glutamyl)DesB30 Human Insulin

15-{3-[2-(2-{3-[3-(2,5-Dioxopyrrolidin-1-yloxycarbonyl)propionylamino]propoxy}ethoxy)ethoxy]-propylcarbamoyl}pentadecanoicacid tert-butyl ester (crude product 0.196 g, 0.264 mmol) reacted withdesB30 insulin (0.132mmol) as described above resulting in 400 mgprecipitate which was Gilson purified, gradient: 0.0-5.0 min 40% A;5.0-15.0 min 40-80% A, fractions at Rt 15.5-16.0 min were collected andevaporated to dryness.

The resulting mass was subsequently treated with TFA/DCM 1/1 (100mL) inorder to deprotect the carboxy groups. After evaporation the resultingproduct was purified 3 times on

Gilson HPLC on a C18 column (Jones, Kromasil RP18 5 μm 15×225 mm).

Gradient: 0.0-5.0 min 35% A; 5.0-20.0 min 20-90%. Fractions from Rt15.0-16.0 min collected and evaporated resulting in 23 mg compound.

MALDI-TOF-MS: m/z 6277.15; calc. 6270.

HPLC (method 5): Rt 9.50 min.

ω-{3-[2-(2-{3-[3-(2,5-Dioxopyrrolidin-1-yloxycarbonyl)propionylamino]propoxy}ethoxy)-ethoxy]propylcarbamoyl}pentadecanoicacid tert-butyl ester

Preparation fromω-[3-(2-{2-[3-(3-carboxypropionylamino)propoxy]ethoxy}ethoxy)propylcarbamoyl-]pentadecanoicacid tert-butyl ester (0.17 g, 0.264 mmol) and TSTU gave 196 mg crudeproduct which was used without further purification. LCMS Method 6: Rt7.36 min; m/z (M+1) 742; Calcd.: 742.

ω-[3-(2-{2-[3-(3-carboxypropionylamino)propoxy]ethoxy}ethoxy)-propylcarbamoyl]pentadecanoicacid tert-butyl ester

Hexadecanedioic acid tert-butyl ester 2,5-dioxo-pyrrolidin-1-yl ester(0.5 g, 1.13 mmol) andN-(3-{2-[2-(3-aminopropoxy)ethoxy]ethoxy}propyl)succinamic acid (0.36 g,1.13 mmol) were reacted as described above.

Purification of the crude product on Gilson HPLC on a C18 column (Jones,Kromasil RP18 5 μm 15×225 mm). Gradient: 0.0-1.0 min 50% A; 1.0-30.0 min50-90%. Fractions with Rt 24.0-26.0 min collecte4d and evaporatedresulting in 170 mg of the target product.

LCMS (Method 6): Rt 7.06 min; m/z (M+1) 645; Calcd.: 645.

Example 19 Synthesis ofN^(εB29)-(3-(3-{4-[3-(ω-Carboxyundecanoylamino)propoxy]butoxypropylcarbamoyl)-propionyl-γ-glutamyl)DesB30 Human Insulin

This compound was prepared similarly as described in example 8 usingdodecanoic acid mono tert-butyl ester.

Data for the title compound:

MALDI-TOF-MS: m/z=6332. Calculated: 6334

HPLC (Method 1): R_(t)=9.57 min.

HPLC (Method 5): R_(t)=7.50 min.

HPLC (Method 6): R_(t)=4.11 min; m/z: 1584 (M+4)/4. Calcd: 1584.

Example 20N^(εB29)-(3-(3-{4-[3-(ω-Carboxytridecanoylamino)propoxy]butoxypropylcarbamoyl)-propionyl-γ-glutamyl)DesB30 Human Insulin

Example 21N^(εB29)-(3-(2-{2-[2-(11-Carboxyundecanoylamino)ethoxy]ethoxy}ethylcarbamoyl)propionyl-γ-glutamyl)DesB30 Human Insulin

Example 22N^(εB29)-(3-(2-{2-[2-(ω-carboxytridecanoylamino)ethoxy]ethoxy}ethylcarbamoyl)propionyl-γ-glutamyl)DesB30 Human Insulin

Example 23N^(εB29)-{3-[2-(2-{2-[2-(ω-Carboxy-pentadecanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]propionyl-gamma-γ-D-glutamyl)DesB30 Human Insulin

Example 24N^(εB29)-{3-[2-(2-{2-[2-(7-carboxyheptanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]propionyl-γ-glutamyl}DesB30 Human Insulin

Example 25N^(εB29)-{3-[2-(2-{2-[2-(9-carboxynonanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]propioniyl-γ-glutamyl}DesB30 Human Insulin

Example 26N^(εB29)-{3-[2-(2-{2-[2-(11-carboxyundecanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]propionyl-γ-glutamyl}DesB30 Human Insulin

Example 27N^(εB29)-{3-[2-(2-{2-[2-(13-carboxytridecanoylamino)ethoxy}ethoxy]ethoxy)ethoxy]propionyl-γ-glutamyl}DesB30 Human Insulin

Example 28 Insulin Receptor Binding of the Insulin Derivatives of theInvention

The affinity of the insulin analogues of the invention for the humaninsulin receptor was determined by a SPA assay (Scintillation ProximityAssay) microtiterplate antibody capture assay. SPA-PVT antibody-bindingbeads, anti-mouse reagent (Amersham Biosciences, Cat No. PRNQ0017) weremixed with 25 ml of binding buffer (100 mM HEPES pH 7.8; 100 mM sodiumchloride, 10 mM MgSO₄, 0.025% Tween-20). Reagent mix for a singlePackard Optiplate (Packard No. 6005190) is composed of 2.4 μl of a1:5000 diluted purified recombinant human insulin receptor-exon 11, anamount of a stock solution of A14 Tyr[¹²⁵I]-human insulin correspondingto 5000 cpm per 100 μl of reagent mix, 12 μl of a 1:1000 dilution of F12antibody, 3 ml of SPA-beads and binding buffer to a total of 12 ml. Atotal of 100 μl was then added and a dilution series is made fromappropriate samples. To the dilution series was then added 100 μl ofreagent mix and the samples were incubated for 16 hours while gentlyshaken. The phases were the then separated by centrifugation for 1 minand the plates counted in a Topcounter. The binding data were fittedusing the nonlinear regression algorithm in the GraphPad Prism 2.01(GraphPad Software, San Diego, Calif.).

Human Serum Albumin Affinity Assay

Relative binding constant of 125I-TyrA14-analogue to human serum albuminimmobilised on Minileak particles and measured at 23° C. (detemir=1 insaline buffer)

Insulin receptor affinity in Albumin affinity in relation to Compoundrelation to human insulin insulin Determir example 1 11 2.381 example 212 10.649 example 3 14 4.273 example 4 9.2 1.201 example 5 5.6 0.48example 6 8.3 3.59 example 7 35 example 8 34 example 9 34 example 10 27example 11 16 example 12 38 example 13 17 example 14 15 example 15 35example 16 31 example 17 17

Example 29 Pulmonary Delivery of Insulin Derivatives to Rats

The test substance will be dosed pulmonary by the drop instillationmethod. In brief, male Wistar rats (app.250 g) are anaesthesized in app.60 ml fentanyl/dehydrodenzperidol/dormicum given as a 6.6 ml/kg scprimingdose and followed by 3 maintainance doses of 3.3 ml/kg sc with aninterval of 30 min. Ten minutes after the induction of anaesthesia,basal samples are obtained from the tail vein (t=−20 min) followed by abasal sample immediately prior to the dosing of test substance (t=0). Att=0, the test substance is dosed intra tracheally into one lung. Aspecial cannula with rounded ending is mounted on a syringe containingthe 200 ul air and test substance (1 ml/kg). Via the orifice, thecannula is introduced into the trachea and is forwarded into one of themain bronchi—just passing the bifurcature. During the insertion, theneck is palpated from the exterior to assure intratracheal positioning.The content of the syringe is injected followed by 2 sec pause.Thereafter, the cannula is slowly drawn back. The rats are keptanaesthesized during the test (blood samples for up to 4 hrs) and areeuthanized after the experiment.

1. An insulin derivatives having a side chain attached either to theα-amino group of the N-terminal amino acid residue of B chain or to anε-amino group of a Lys residue present in the B chain of the parentinsulin molecule via an amide bond wherein said side chain comprises oneor more residues of ethylenglycol, propyleneglycol and/or butyleneglycolcontaining independently at each termini a group selected from —NH₂ and—COOH; a fatty diacid moiety with from 4 to 22 carbon atoms, at leastone free carboxylic acid group or a group which is negatively charged atneutral pH; and possible linkers which link the individual components inthe side chain together via amide or ether bonds, said linkersoptionally comprising a free carboxylic acid group.
 2. The insulinderivative according to claim 1, wherein PEG or PPG or PBG group hasfrom 2 to 20; from 2 to 10 or from 2 to 5 residues of ethyleneglycol,propyleneglycol or butyleneglycol.
 3. The insulin derivative accordingto claim 1, wherein the sidechain comprises a single residue ofethyleneglycol.
 4. The insulin derivative according to claim 1, whereinthe sidechain comprises single residues of ethylenglycol,propyleneglycol and butyleneglycol alone or in combination.
 5. Theinsulin derivative according to claim 4, wherein the sidechain comprisesone residue of propyleneglycol and one residue of butyleneglycol.
 6. Theinsulin derivative according to claim 1, wherein the fatty diacidcomprises from 4 to 22 carbon atoms in the carbon chain.
 7. The insulinderivative according to claim 6, wherein the fatty diacid comprises from6 to 22, from 8 to 20, from 8 to 18, from 4 to 18, from 6 to 18, from 8to 16, from 8 to 22, from 8 to 17 or from 8 to 15 carbon atoms in thecarbon chain.
 8. The insulin derivative according to claim 1, whereinthe linker is an amino acid residue, a peptide chain of 2-4 amino acidresidues or has the motif α-Asp, β-Asp, α-Glu, γ-Glu, α-hGlu, δ-gGlu,—N(CH₂COOH)CH₂CO—, —N(CH₂CH₂COOH)CH₂CH₂CO—, —N(CH₂COOH)CH₂CH₂CO— or—N(CH₂CH₂COOH)CH₂CO—
 9. The insulin derivative according to claim 1,wherein the Lys residue in the B chain of the parent insulin in eitherposition B3, B29 or in one of positions B23-30.
 10. The insulinderivative according to claim 1 having the formula

wherein Ins is the parent insulin moiety which via the α-amino group ofthe N-terminal amino acid residue of the B chain or an ε-amino group ofa Lys residue present in the B chain of the insulin moiety is bound tothe CO— group in the side chain via an amide bond; each n isindependently 0, 1, 2, 3, 4, 5 or 6; Q₁, Q₂, Q₃, and Q₄ independently ofeach other can be selected from: (CH₂CH₂O)_(s)—; (CH₂CH₂CH₂O)_(s)—;(CH₂CH₂CH₂CH₂O)_(s)—; (CH₂CH₂OCH₂CH₂CH₂CH₂O)_(s)— or(CH₂CH₂CH₂OCH₂CH₂CH₂CH₂O)_(s)— where s is 1-20; —(CH₂)_(r)— where r isan integer from 4 to 22; or a divalent hydrocarbon chain comprising 1, 2or 3 —CH═CH— groups and a number of —CH₂— groups sufficient to give atotal number of carbon atoms in the chain in the range of 4 to 22;—(CH₂)_(t)— or —(CH₂OCH₂)_(t)—, where t is an integer from 1 to 6;—(CR₁R₂)_(q)—, where R₁ and R₂ independently of each other can be H,—COOH, (CH₂)₁₋₆COOH and R₁ and R₂ can be different at each carbon, and qis 1-6; —((CR₃R₄)_(q1))₁—(NHCO—(CR₃R₄)_(q1)—NHCO)₁₋₂—((CR₃R₄)_(q1))₁ or—((CR₃R₄)_(q1))₁—(CONH—(CR₃R₄)_(q1)—CONH)₁₋₂—((CR₃R₄)_(q1)—)—,—((CR₃R₄)_(q1))₁—(NHCO—(CR₃R₄)_(q1)—CONH)₁₋₂—((CR₃R₄)_(q1))₁ or—((CR₃R₄)_(q1))₁—(CONH—(CR₃R₄)_(q1)—NHCO)₁₋₂—((CR₃R₄)_(q1))₁ where R₃and R₄ independently of each other can be H, —COOH, and R₃ and R₄ can bedifferent at each carbon, and q₁ is 1-6-; and a bond; with the provisothat Q₁-Q₄ are different; X₁, X₂ and X₃ are independently selected from:O; a bond;

where R is hydrogen or —(CH₂)_(p)—COOH, —(CH₂)_(p)—SO₃H,—(CH₂)_(p)—PO₃H₂, —(CH₂)_(p)—O—SO₃H; —(CH₂)_(p)—O—PO₃H₂; or—(CH₂)_(p)-tetrazol-5-yl, where each p independently of the other p's isan integer in the range of 1 to 6; and Z is selected from: —COOH;—CO-Asp; —CO-Glu; —CO-Gly; —CO-Sar; —CH(COOH)_(2;) —N(CH₂COOH)₂; —SO₃H;—OSO₃H; —OPO3H₂, —PO₃H₂ and -tetrazol-5-yl and any Zn²⁺ complex thereof.11. The insulin derivative according to claim 10, wherein s is in therange of 2-12, 2-4 or 2-3
 12. The insulin derivative according to claim10, wherein s is preferably
 1. 13. The insulin derivative according toclaim 10, wherein Z is —COOH.
 14. The insulin derivative according toclaim 1, wherein the parent insulin is a desB30 human insulin analogue.15. The insulin derivative according to claim 1, wherein the parentinsulin is selected from the group consisting of human insulin; desB1human insulin; desB30 human insulin; GlyA21 human insulin; GlyA21 desB30human insulin; AspB28 human insulin; porcine insulin; LysB28 ProB29human insulin; GlyA21 ArgB31 ArgB32 human insulin; and LysB3 GIuB29human insulin or AspB28 desB30 human insulin.
 16. The insulin derivativeaccording to claim 1 selected from the group consisting ofN^(εB29)-(3-[2-{2-(2-[ω-carboxy-pentadecanoyl-γ-glutamyl-(2-amino-ethoxy)]-ethoxy)-ethoxy}-ethoxy]-propinoyl)desB30 human insulin,N^(εB29)-(3-[2-{2-(2-[ω-carboxy-heptadecanoyl-γ-glutamyl-(2-amino-ethoxy)]-ethoxy)-ethoxy}-ethoxy]-propinoyl)desB30 human insulin,N^(εB29)-{3-[2-(2-{2-[2-(ω-carboxy-pentadecanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionyl-γ-glutamyldesB30 human insulin,N^(εB29)-(ω-[2-(2-{2-[2-(2-carboxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethylcarbamoyl]-heptadecanoyl-α-glutamyl)desB30 human insulin,N^(εB29)-(ω-[2-(2-{2-[2-(2-carboxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethylcarbamoyl]-heptadecanoyl-γ-glutamyl)desB30 human insulin,N^(εB29)-3-[2-(2-{2-[2-(ω-carboxy-heptadecanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionyl-γ-glutamyldesB30 human insulin,N^(εB29)-(3-(3-{2-[2-(3-[7-carboxyheptanoylamino]propoxy)ethoxy]-ethoxy}propylcarbamoyl)propionyl)desB30 human insulin,N^(εB29)-(3-(3-{4-[3-(7-Carboxyheptanoylamino)propoxy]butoxy}propylcarbamoyl)-propionyl-γ-glutamyl)desB30 human insulin,N^(εB29)-(3-(3-{2-[2-(3-[9-Carboxynonanoylamino]propoxy)ethoxy]ethoxy}-propylcarbamoyl)propionyl)desB30 human insulin,N^(εB29)-(3-(2-{2-[2-(9-carboxynonanoylamino)ethoxy]ethoxy}ethylcarbamoyl)propionyl-γ-glutamyl)desB30 human insulin,N^(εB29)-(3-(3-{4-[3-(9-Carboxynonanoylamino)propoxy]butoxy}-propylcarbamoyl)propionyl-γ-glutamyl)desB30 human insulin,N^(εB29)-(2-[3-(2-(2-{2-(7-carboxyheptanoylamino)ethoxy}ethoxy)-ethylcarbamoyl]propionyl-γ-glutamyl)desB30 human insulin,N^(εB29)-(3-[2-(2-{2-[2-(ω-carboxypentadecanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]propionyl))desB30 human insulin,N^(εB29)-(3-(2-{2-[2-(2-{2-[2-(2-{2-[2-(2-{2-[2-(ω-carboxy-tridecanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionoyl-γ-glutamyl)desB30 human insulin,N^(εB29)'-(3-[2-(2-{2-[2-(ω-Carboxy-tridecanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionoyl-γ-glutamyl)desB30 human insulin,N^(εB29)-(3-[2-(2-{2-[2-(2-{2-[2-(ω-carboxy-tridecanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-propionoyl-γ-glutamyl)desB30 human insulin,N^(εB29)-(3-(2-{2-[2-(ω-Carboxy-pentadecanoylamino)-ethoxy]-ethoxy}-ethylcarbamoyl)-propionyl-γ-glutamyl)desB30 human insulin,N^(εB29)-(3-(3-{2-[2-(3-[ω-Carboxypentadecanoylamino]propoxy)ethoxy]-ethoxy}propylcarbamoyl)propionyl-γ-glutamyl)desB30 human insulin,N^(εB29)-(3-(3-{4-[3-(ω-Carboxyundecanoylamino)propoxy]butoxypropylcarbamoyl)propionyl-γ-glutamyl)desB30 human insulin,N^(εB29)-(3-(3-{4-[3-(ω-carboxytridecanoylamino)propoxy]butoxypropyl-carbamoyl)propionyl-γ-glutamyl)desB30 human insulin,N^(εB29)-(3-(2-{2-[2-(ω-Carboxyundecanoylamino)ethoxy]ethoxy}ethylcarbamoyl)propionyl-γ-glutamyl)desB30 human insulin,N^(εB29)-(3-(2-{2-[2-(ω-carboxytridecanoylamino)ethoxy]ethoxy}ethylcarbamoyl)-propionyl-γ-glutamyl)desB30 human insulin,N^(εB29)-{3-[2-(2-{2-[2-(ω-carboxy-pentadecanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]propionyl-gamma-γ-D-glutamyl)desB30 human insulin,N^(εB28)-{3-[2-(2-{2-[2-(7-carboxyheptanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]-propionyl-γ-glutamyl}desB30 human insulin,N^(εB29)-{3-[2-(2-{2-[2-(9-carboxynonanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]propioniyl-γ-glutamylldesB30 human insulin,N^(εB29)-{3-[2-(2-{2-[2-(ω-carboxyundecanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]-propionyl-γ-glutamyl}desB30 human insulin,N^(εB29)-{3-[2-(2-{2-[2-(ω-carboxytridecanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]propionyl-γ-glutamyl}desB30 human insulin.
 17. A method of treating diabetes in a patient inneed of such a treatment, comprising administering to the patient atherapeutically effective amount of an insulin derivative, said insulinderivative having a side chain attached either to the α-amino group ofthe N-terminal amino acid residue of B chain or to an s-amino group of aLys residue present in the B chain of the parent insulin molecule via anamide bond which side chain comprises one or more residues ofethylenglycol, propyleneglycol and/or butyleneglycol containingindependently at each termini a group selected from —NH₂ and —COOH; afatty diacid moiety with from 4 to 22 carbon atoms, at least one freecarboxylic acid group or a group which is negatively charged at neutralpH; and possible linkers which link the individual components in theside chain together via amide or ether bonds, said linkers optionallycomprising a free carboxylic acid group together with a pharmaceuticallyacceptable carrier.
 18. A method of treating diabetes in a patient inneed of such a treatment, comprising administering to the patient atherapeutically effective amount of an insulin derivative, said insulinderivative having a side chain attached either to the α-amino group ofthe N-terminal amino acid residue of B chain or to an s-amino group of aLys residue present in the B chain of the parent insulin molecule via anamide bond which side chain comprises one or more residues ofethylenglycol, propyleneglycol and/or butyleneglycol containingindependently at each termini a group selected from —NH₂ and —COOH; afatty diacid moiety with from 4 to 22 carbon atoms, at least one freecarboxylic acid group or a group which is negatively charged at neutralpH; and possible linkers which link the individual components in theside chain together via amide or ether bonds, said linkers optionallycomprising a free carboxylic acid group in mixture with an insulin or aninsulin analogue which has a rapid onset of action, together with apharmaceutically acceptable carrier.